InteractiveFly: GeneBrief

spenito: Biological Overview | References

BIOLOGICAL OVERVIEW

N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m(6)A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. This study identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m(6)A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m(6)A levels and is involved in sex determination in Drosophila. Flacc promotes m(6)A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, this work advances the molecular understanding of conservation and regulation of the m(6)A machinery (Knuckles, 2018).

In the past years, N⁶-methyladenosine (m⁶A) RNA has emerged as an abundant and dynamically regulated modification throughout the transcriptome. m⁶A affects almost every stage of mRNA metabolism, and its absence is associated with various defects in meiosis, embryonic stem cell (ESC) differentiation, DNA repair, circadian rhythm, neurogenesis, dosage compensation, and sex determination. Alteration of m⁶A levels also promotes glioblastoma progression and is linked to poor prognosis in myeloid leukemia (Knuckles, 2018).

Formation of m⁶A is catalyzed by the activity of methyltransferase-like 3 (METTL3; also called MT-A70), which physically interacts with METTL14, Wilms tumor 1-associated protein (WTAP), Vir-like m⁶A methyltransferase-associated (KIAA1429/VIRMA), and RNA-binding motif 15 (RBM15) and its paralog, RBM15B. Drosophila has corresponding homologs Mettl3, Mettl14, Fl(2)d, Virilizer (Vir), and Spenito (Nito) (Lence, 2017). Recent crystal structural studies investigated the molecular activities of the two predicted methyltransferases METTL3 and METTL14. Only METTL3 was shown to contain the catalytic activity and form a stable heterodimer with METTL14, which was required to enhance METTL3 enzymatic activity by binding substrate RNA and positioning the methyl group for transfer to adenosine. In addition, WTAP [Fl(2)d] ensures the stability and localization of the heterodimer to nuclear speckles (Lence, 2016). VIRMA (Vir) is essential for m⁶A deposition, but its molecular function is currently unknown. Last, RBM15 and RBM15B (Nito) have been suggested to recruit the methyltransferase complex to its target transcripts via direct binding to U-rich sequences on mRNA. In humans, this function is important to control m⁶A promoted X-chromosome inactivation via XIST-mediated transcriptional repression. In Drosophila, Nito promotes m⁶A function in the sex determination and dosage compensation pathways (Lence, 2016; Knuckles, 2018 and references therein).

To date, it is unknown how Nito in Drosophila interacts with other members of the methyltransferase writer complex to ensure their recruitment to mRNA targets. Although, in human cells, RBM15/15B were reported to interact with METTL3 in a WTAP-dependent manner, it is unclear whether this interaction is conserved in other organisms. In order to address these questions, interactome analyses from Mus musculus and Drosophila melanogaster cell extracts were performed using Rbm15 and Nito as bait, respectively. Mouse zinc finger CCCH domain-containing protein 13 (Zc3h13) and its fly homolog, CG7358, which was named Fl(2)d-associated complex component (Flacc), as novel interactors of the m⁶A writer machinery. A lack of these proteins dramatically reduces m⁶A levels in both organisms. Consistent with the role of m⁶A in sex determination in Drosophila, Flacc depletion results in aberrant splicing of Sex lethal (Sxl) and leads to transformations of female into male-like structures. Moreover, it was demonstrated that Flacc interacts with Nito and Fl(2)d and serves as an adaptor between these two proteins, thereby stabilizing the complex and promoting m⁶A deposition on mRNA (Knuckles, 2018).

This study has identified a novel interactor of the m⁶A methyltransferase complex, which is conserved in Drosophila and mice. Its function in the m⁶A pathway is essential in both species, as its absence results in dramatic reduction of m⁶A levels. The facts that the human homolog was found recently in interactome studies with WTAP and that it can rescue the interaction between Fl(2)d and Nito in Drosophila suggest that it has a similar role in human cells. Despite this functional conservation, the protein sequence identity among different homologs is rather weak. Mouse Zc3h13 contains several additional domains as compared with Flacc. In particular, it differs by the presence of a zinc finger domain, which is present in a common ancestor but was lost in dipterian. Other species such as Ciona intestinalis also lack the zinc finger motif. In addition, the zinc finger motif can be found in two variants across evolution: one short and one long. As zinc finger motifs are commonly involved in nucleic acid binding or protein-protein interactions, it will be interesting to address the functional importance of this domain when present in the protein. Of note, Zc3h13 appears completely absent in nematodes, as is also the case for Mettl3, possibly indicating that these two proteins have coevolved for the regulation of adenosine methylation (Knuckles, 2018).

The current work strongly supports the existence of at least two distinct stable complexes that interact weakly to regulate m⁶A biogenesis. This result is consistent with earlier studies by Rottman and colleagues (Bokar, 1997), who isolated two protein components using an in vitro methylation assay and HeLa cell nuclear extracts, which are readily dissociable under nondenaturing conditions. Gel filtration and gradient glycerol sedimentation estimated molecular weights of 200 and 875 kDa. While biochemical characterization will be required to address the exact identity of the different complex components, recent biochemical analysis suggests that the 200-kDa complex consists of Mettl3 and Mettl14 (Liu, 2014). Although the exact composition of the larger complex is currently unknown, it is postulated that it is probably MACOM, consisting of Wtap, Virma, Hakai, Rbm15, and Zc3h13. The calculated total molecular weight of these proteins (600 kDa) is lower than that of the large complex (875 kDa), which suggests the presence of other factors or the inclusion of some subunits in multiple copies. For instance, recombinant WTAP can form aggregates, suggesting the possibility of higher complex organization (Liu, 2014). Finally, the existence of two complexes is also supported by a genetic analyses, which show that the knockout of Mettl3 and Mettl14 results in viable animals, while loss of function of fl(2)d, vir, nito, and flacc is lethal during development. This indicates that the MACOM acts beyond m⁶A methylation via Mettl3 (Knuckles, 2018).

m6A modulates neuronal functions and sex determination in Drosophila

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. This study carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. The member of the split ends protein family, Spenito, was identified as a novel bona fide subunit of the methyltransferase complex. Important roles of this complex were demonstrated in neuronal functions and sex determination, and the nuclear YT521-B protein was implicated as a main m6A effector in these processes. Altogether, this work substantially extends knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal (Lence, 2016).

Components of the methyltransferase complex have been shown to be essential during early development of various organisms. In contrast to these studies, the current analysis argues against a vital role for Ime4 in Drosophila as both deletion alleles give rise to homozygous adults without prominent lethality during development. This cannot be explained by compensation via dMettl14, as its knockout produces similar effects as the Ime4 (Methyltransferase like 3) knockout. Furthermore, depleting both genes only slightly intensifies Mthe locomotion phenotype without affecting fly survival, supporting the idea that Ime4 and dMettl14 act together to regulate the same target genes. Accordingly, loss of either component in vivo dramatically affects stability of the other (Lence, 2016).

Loss of function of either of the methyltransferases produces severe behavioural defects. All of them can be rescued by specific expression of Ime4 cDNA in the nervous system of Ime4 mutants, indicating neuronal functions. This is consistent with the substantial enrichment of m6A and its writer proteins in the embryonic neuroectoderm, as well as with the affected genes upon depletion in S2R+ cells. These analyses further reveal notable changes in the architecture of NMJs, potentially explaining the locomotion phenotype. In the mouse, m6A is enriched in the adult brain, whereas in zebrafish, METTL3 and WTAP show high expression in the brain region of the developing embryo. Furthermore, a crucial role for the mouse m6A demethylase FTO in the regulation of the dopaminergic pathway was clearly demonstrated. Thus, together with previous studies, this work reveals that m6A RNA methylation is a conserved mechanism of neuronal mRNA regulation contributing to brain function (Lence, 2016).

This study found that Ime4 and dMettl14 also control the splicing of the Sxl transcript, encoding for the master regulator of sex determination in Drosophila. This is in agreement with the previously demonstrated roles of Fl(2)d and Vir in this process. However, in contrast to these mutants, mutants for Ime4, dMettl14 and YT521-B are mostly viable, ruling out an essential role in sex determination and dosage compensation. Only when one copy of Sxl is removed, Ime4 mutant females start to die. Notably, m6A effect on Sxl appears more important in the brain compared to the rest of the organism, possibly allowing fly survival in the absence of this modification (Lence, 2016).

A targeted screen identifies Nito as a bona fide methlytransferase complex subunit. The vertebrate homologue of Spenito, RBM15, was recently shown to affect XIST gene silencing via recruitment of the methyltransferase complex to XIST RNA, indicating that its role in m6A function and dosage compensation is conserved. In summary, this study provides a comprehensive in vivo characterization of m6A biogenesis and function in Drosophila, demonstrating the crucial importance of the methyltransferase complex in controlling neuronal functions and fine-tuning sex determination via its nuclear reader YT521-B (Lence, 2016).

An autonomous metabolic role for Spen

Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. This study found that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in beta-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction (Hazegh, 2017).

This work provides the first detailed investigation of a fat regulatory role for Spen in any organism, and the first evidence that Nito also functions in this process. Spen depletion in the fat body drastically increased stored fat. Spen has been implicated in multiple pathways involved in endocrine signaling, including Notch, Wingless, and nuclear receptor signaling. This study found it unlikely that nuclear receptor pathways are relevant to the fat regulatory role this study defines, because upon Spen depletion or overexpression consistent changes in the expression of genes that are targets of those pathways were not observed. Furthermore, the lack of phenotypes involving fat storage per se upon overexpression of C-terminal Spen-SPOC^only domain argues against a role for Wg signaling, in which the same construct has potent dominant negative effects. Conversely, whereas a C-terminally truncated version of mSpen has little effect on Notch signaling, the strong fat phenotypes resulting from Spen-ΔSPOC overexpression suggest that Spen does not regulate fat via the Notch pathway (Hazegh, 2017).

Notably, Spen KD larvae also exhibited behavioral changes (increased food intake, decreased locomotion) that may have contributed to the fat increase. Thus, in addition to direct roles in fat accumulation within fat storage cells, Spen may be involved in a cross-talk pathway between the FB and the brain. However, a model is strongly support wherein increased food intake is instead an attempt to compensate for a condition of 'perceived starvation' resulting from an inability to access energy stores. Similarly, a lack of available energy may restrict locomotion. This hypothesis is further strengthened by the observation that Spen overexpression was sufficient to deplete stored fat but did not cause opposing behavioral phenotypes (Hazegh, 2017).

Mosaic analysis confirmed an autonomous role for Spen in FB cells. Spen KD in clones throughout the FB showed a striking increase in LD size. Larger LDs normally have lower surface tension, and the stored fat is easier to access. LD remodeling in WT animals is a highly regulated process involving specific factors, some of which were identified in a genome-wide RNAi screen in cultured Drosophila S2 cells. Notably, RNAseq data revealed that the products of several LD-regulating genes were significantly altered by Spen depletion, including l(2)01289 (~7-fold decreased), CG3887 (1.3-fold decreased), and eIF3-S9 (1.5-fold increased). While it is unclear if these changes are direct effects of Spen depletion, they may explain why LDs in Spen KD larvae are large yet apparently inaccessible, resulting in starvation sensitivity (Hazegh, 2017).

Consistent with the observed changes in FB cell and LD morphology and starvation sensitivity, changes in metabolites and gene expression in Spen KD larvae pointed to a drastic defect in lipid catabolism. Defects in β-oxidation were the most obvious, in part because the opposite effects were observed upon FB-restricted Spen overexpression. Spen depletion led to a decrease in the levels of free and acyl-conjugated carnitine, as well as of transcripts of three of the four enzymes necessary to break down acyl-carnitines into free fatty acids. Three lipases were also downregulated, which likely further contributes to an inability to convert energy stored as TAGs into usable forms. While an apparent upregulation of gluconeogenesis is evident, as supported by alterations in aspartate and PEPCK expression, these processes may be unable to completely compensate for decreased trehalose utilization, and these defects may contribute to the lethargy phenotype resulting from Spen KD. Consequently, surviving the loss of Spen may require breakdown of protein into free amino acids in order to anaplerotically replenish the TCA cycle, consistent with changes in expression of proteases, the observed decrease in many free amino acids, as well as increases in protein catabolism and collagen turnover markers (N-acetylmethionine and hydroxyproline). Of note, sustained proteolysis is a marker of aging and inflammation, a phenotype that has been previously associated with decreased locomotion in human and mouse models of physical activity, suggesting potential future ramifications of Spen’s role in metabolism with respect to aging/inflammation research. Finally, the observed decrease in glycogen levels upon Spen KD supports a model wherein glycogen is used as a carbohydrate source (in lieu of decreased levels of trehalose) to fuel glycolysis. The overall metabolic defects described in this study are distinctly different from what has been observed upon manipulation of other fat regulators (e.g., Sir2), suggesting that Spen operates in a previously undescribed pathway (Hazegh, 2017).

The results with Spen and Nito truncations provide additional mechanistic insight into how these proteins function in fat regulation. Overexpressing Spen-ΔSPOC reversed the phenotype of full-length Spen overexpression, and instead resulted in similar phenotypes to Spen depletion. Nito-ΔC overexpression had the same effects: larvae arrested development and FB clones mimicked starvation even when dietary nutrients were abundant. Overexpression of the Spen-SPOC^only construct had no effect on FB cells, as was the case for Nito-ΔN. Thus only Spen harboring the RRMs and the SPOC domain was able to promote fat depletion when overexpressed. Conversely, only truncated forms of Spen or Nito that retain the RRMs dominantly perturbed both FB cell viability and organismal resistance to starvation (Hazegh, 2017).

Recent studies of X chromosome inactivation found that mSpen RRMs mediate binding to the lncRNA Xist. Rbm15 (mNito) also binds Xist, and is required for N⁶-methyladenosine (m⁶A) modification of that lncRNA, which is in turn required for its ability to repress X chromosome transcription. Nito is a subunit of the Drosophila m⁶A methyltransferase complex and is required for RNA binding by that complex; Nito knockdown severely decreases global m⁶A modification of mRNA (Lence, 2016). Interestingly, the m⁶A demethylase FTO/ALKBH9 was the first human obesity susceptibility gene identified by genome-wide association studies, but the relevant nucleic acid target(s) remain unknown. This work provides the first hint that an RNA bound by Spen and/or Nito may be a key FTO substrate (Hazegh, 2017).

These findings lead to a model for Spen and Nito function in the regulation of fat storage. Spen binds via its RRMs to one or more RNAs and, via recruitment of other factors, promotes the expression of enzymes key for mobilization of energy stored as fat (e.g. lipases). The mechanism of activation may be direct or indirect, and via alternative splicing, activation/repression of transcription, or effects on RNA stability and/or translation. Moreover, RNA binding partners may be mRNA or non-coding RNA. Future work will be required to make these distinctions. It is proposed that the Spen SPOC domain is critical for this function, but undefined domains in between the N-terminal RRMs and C-terminal SPOC domain are also important, and these are not shared with Nito. It is proposed that Nito binds via its RRMs the same or a largely overlapping set of RNAs, and also recruits additional factors via its SPOC domains, but–either because it fails to recruit specific factors recruited by Spen, or because it recruits other factors not recruited by Spen-Nito ultimately inhibits/represses the same energy-storage-mobilizing enzymes that are activated by Spen. Overexpressed Spen or Nito fragments containing RRMs sequester target RNAs away from endogenous full-length Spen and the other effectors of fat storage control. Finally, the findings in mouse adipose tissue that mSpen and mNito both increase in expression when a HFD drives fat accumulation lead to the belief that in WT animals Nito acts as a counterbalance to Spen in order to fine-tune fat storage. Future studies delving into more mechanistic details may lead to treatments for obesity and related metabolic disorders that result from perturbation of the pathway that was elucidated here (Hazegh, 2017).

The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila

Split ends (SPEN) is the founding member of a well conserved family of nuclear proteins with critical functions in transcriptional regulation and the post-transcriptional processing and nuclear export of transcripts. In animals, the SPEN proteins fall into two size classes that perform either complementary or antagonistic functions in different cellular contexts. This study shows that the two Drosophila representatives of this family, SPEN and Spenito (NITO), regulate metamorphic remodeling of the CCAP/bursicon neurosecretory cells. CCAP/bursicon cell-targeted overexpression of SPEN had no effect on the larval morphology or the pruning back of the CCAP/bursicon cell axons at the onset of metamorphosis. During the subsequent outgrowth phase of metamorphic remodeling, overexpression of either SPEN or NITO strongly inhibited axon extension, axon branching, peripheral neuropeptide accumulation, and soma growth. Cell-targeted loss-of-function alleles for both spen and nito caused similar reductions in axon outgrowth, indicating that the absolute levels of SPEN and NITO activity are critical to support the developmental plasticity of these neurons. Although nito RNAi did not affect SPEN protein levels, the phenotypes produced by SPEN overexpression were suppressed by nito RNAi. It is proposed that SPEN and NITO function additively or synergistically in the CCAP/bursicon neurons to regulate multiple aspects of neurite outgrowth during metamorphic remodeling (Gu, 2017).

The SPEN family has been evolutionarily conserved, with representatives from protists and plants to animals. The family includes mouse MINT (Msx2-interacting nuclear target protein) and human SHARP (SMRT/HDAC1 associated repressor protein). SPEN, SHARP, and MINT are unusually large proteins of ~3575 to 5500 amino acids in length. In addition to SPEN, the Drosophila genome contains one other SPEN-like gene, spenito (nito), that encodes a much smaller, 793 amino acid protein . Although SPEN and NITO share conserved N-terminal RRMs and the SPOC domain, their overall sequence similarity is only 28%, suggesting that SPEN and NITO may function similarly in some processes, but differently in others. For example, SPEN and NITO display functional antagonism during eye development (Jemc, 2006) but act synergistically in regulating Wingless signaling in wing imaginal discs and cultured Kc cells (Chang, 2008). In addition, NITO was identified as a splicing factor involved in Sxl regulation and sex determination in Drosophila, while SPEN had no such effect (Yan, 2015). Several studies in vertebrates also suggest that the relationship between these two proteins is context-dependent (Chang, 2008). Similar to SPEN, NITO is broadly expressed in Drosophila tissues (Chang, 2008). Therefore, this study investigated the function of NITO and its possible interactions with SPEN in the context of remodeling of the CCAP/bursicon neurons (Gu, 2017).

Several previous studies have examined the role of SPEN during neuronal differentiation. In embryos, SPEN contributes to neuronal cell fate specification and regulates the growth, pathfinding, and fasciculation of PNS and CNS axons. SPEN also regulates proliferation and differentiation of Drosophila photoreceptor neurons. In addition to its role in neuronal differentiation, SPEN has also been shown to modify late-onset, progressive neurodegeneration in a model for spinocerebellar ataxia in the mature Drosophila retina. This study reports that SPEN regulates developmental plasticity in mature neurons (Gu, 2017).

SPEN overexpression specifically inhibited neurite outgrowth during metamorphic remodeling of the CCAP/bursicon cells, with little or no effect on the larval morphology of these neurons. The reasons for the stage-dependence of SPEN activity in these cells are unknown, but one intriguing possibility is that the stage-dependence results from a direct or indirect link to the ecdysone titer during metamorphosis. This is suggested by work on the human SPEN ortholog, SHARP. SHARP expression is steroid-inducible. In addition, the RRM domains of SHARP interact directly with the steroid receptor RNA cofactor SRA, which acts as a scaffold to bring together nuclear receptors, corepressors, and coactivators. SHARP inhibits the transcriptional activity of SRA-stimulated estrogen and glucocorticoid receptors. No Drosophila ortholog of SRA has yet been identified. Nevertheless, in future studies, it will be of interest to examine interactions between SPEN and signaling by the ecdysone receptor (EcR) during neuronal remodeling. (Gu, 2017).

The overexpression of spen and nito produced axon growth and branching phenotypesthat were qualitatively similar (although not identical) to the loss-of-function effects of both genes. Such similarities in GOF and LOF phenotypes are often observed in systems where the stoichiometric ratio of gene products to the concentration of other cellular components is important. Thus, the current observations indicate that SPEN may need to be maintained at a specific level, or within an expression window, to properly regulate axonal outgrowth. Recent studies observed strong genetic interactions between spen and multiple factors controlling Myosin II activity. Notably, many cellular movements, including growth cone migration and branching, depend upon dynamic cytoskeletal rearrangements that can be disrupted by either increasing or decreasing the stability or function of cytoskeletal components. Thus, the interaction between SPEN and Myosin II provides one possible explanation for the similar responses of the CCAP/bursicon neurons to reduced versus enhanced levels of SPEN activity. Interestingly, SPEN also interacts genetically with crinkled (Myosin VIIA) in controlling wing vein development and wing bristle positioning, suggesting a more general function of SPEN in cytoskeletal rearrangements. If SPEN and NITO regulate common cellular processes, then this explanation may also apply to NITO (Gu, 2017).

The current results showed that NITO and SPEN act additively or synergistically to regulate CCAP/bursicon cell remodeling. This finding is in agreement with other studies showing that SPEN and NITO are both positive regulators of Wg signaling in developing wing discs and Drosophila Kc cells, and of programmed cell death in the eye disc induced by the pro-apoptotic factors Head involution defective and Reaper. In contrast, SPEN and NITO are antagonistic in regulating photoreceptor number, rhabdomere morphology, and regular ommatidial spacing in the adult eye (Jemc, 2006). Furthermore, SPEN and NITO may play completely different functions in some situations, as NITO regulates Sxl level and its alternative splicing, while SPEN has no such effects (Yan, 2015). Thus, the interaction between SPEN and NITO depends on the cellular context. In mammals, the large and small SPEN-like proteins display similar context-dependence in the regulation of Notch signaling. The large SPEN family proteins MINT and SHARP both suppress Notch signaling by competing with the N intracellular domain (NICD) for binding to the core transcription factor RBP-J. The NITO ortholog, RBM15, also complexes with RBP-J, but it either stimulates or represses expression of a reporter in different cell lines. Therefore, in Drosophila and in vertebrates, large and small SPEN family proteins act redundantly or synergistically in some cellular contexts and antagonistically in others, or they perform completely different roles (Gu, 2017).

Recent insights into the molecular interactions with SPEN family proteins provide some clues to mechanisms underlying these context-dependent differences in function. In different systems, the large SPEN family proteins function as either transcriptional corepressors or coactivators. For example, human SHARP and RBM15 were identified as crucial factors required for the long non-coding RNA Xist-mediated silencing of X-chromosomes by directly interacting with the Xist, recruiting nuclear corepressor, SMRT, activating histone deacetylase3 (HDAC3), and deacetylating histones to exclude Pol II and repress transcription. Other studies have revealed functions of the SPEN proteins in transcription activation. MINT enhances transcriptional activation at the osteocalcin promoter and associates with an actively phosphorylated and processive form of RNA polymerase II. SHARP enhances beta-catenin/T cell factor (TCF)-mediated transcription. In differentiating Drosophila hemocytes, SPEN binds to many target gene promoters in association with a known activating histone modification pattern (Gu, 2017).

The small SPEN proteins have not been shown to function as transcriptional coactivators or corepressors and instead play important roles in alternative splicing, selection of alternative polyadenylation sites, and nuclear export of mRNAs. However, both the large and small SPEN proteins may function together in protein complexes that first associate with nascent transcripts. These messenger ribonucleoprotein (mRNP) complexes are dynamic structures that participate in transcription, pre-mRNA splicing, and nuclear export, and they change in protein composition to permit nuclear export once pre-mRNAs are completely spliced. Importantly, several studies have detected associations among the large and small SPEN proteins in these structures . Given the strong evolutionary conservation of the large and small SPEN proteins, similar interactions are likely to explain some of the overlapping functions of SPEN and NITO during neuronal remodeling in Drosophila (Gu, 2017).

spenito is required for sex determination in Drosophila melanogaster

Sex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. This study identifies a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. It was shown that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, these results suggest that these factors might be implicated in tumorigenesis through splicing regulation (Yan, 2015).

This study describes the characterization of Nito as a novel component of the Drosophila sex determination pathway. Nito loss-of-function results in stem-cell tumor phenotypes in the germ-line and sexual transformations in the soma. Interestingly, Nito affects Sxl protein levels in both GSCs and somatic tissues by regulating Sxl pre-mRNA alternative splicing, most likely directly as Nito interacts with the Sxl protein and pre-mRNA. The role of Nito is reminiscent of the previously reported roles of splicing factors in Sxl auto regulation, such as both subunits of U2AF, Fl(2)d, SPF45, Vir, and Snf. These data support earlier reports that Sxl physically interacts with components of the spliceosome to simultaneously block utilization of the 3' and 5' splice sites of the male exon (Yan, 2015).

Nito and Spen are members of the SPEN protein family that are evolutionarily conserved from plants, worms, flies to mice and humans. Both proteins contain three N-terminal RRM domains and one C-terminal SPOC domain. The sequence similarity between these domains is low and there is no conservation outside these motifs, suggesting that they have evolved specific functions following a duplication event, as indicated by the observation that spen is not required for Sxl regulation. In Drosophila, spen was first identified in several genetic screens looking for components of the receptor tyrosine kinase (RTK) signaling pathway. Subsequent studies found that spen is implicated in a variety of cellular and developmental processes including neuronal cell fate specification, axon guidance, cell cycle, Hox gene regulation, and cell death. These pleiotropic effects are likely due to the involvement of spen in multiple signaling pathways. However, the molecular mechanisms underlying the function of Spen in these pathways are not understood (Yan, 2015).

Genetic studies in Drosophila have shown that nito overexpression results in a rough eye phenotype and that it plays a redundant role with spen in Wnt signaling, but how Nito is involved in these processes is not known. Biochemical studies indicate that Nito, like its human ortholog, copurify with the precatalytic spliceosome (complex B). In addition, nito, as well as many other splicing factors, was identified in an RNAi screen for RAS/MAPK signaling components. Consistent with these findings, this study found that nito is required for the alternative splicing of the master sex-determination gene Sxl. Previously, both Spen and Nito were thought to act mainly as transcription factors through their SPOC domains, the current findings however clearly indicate that Nito is involved in mRNA splicing. It is intriguing to note that PPS, another important factor required for Sxl splicing, also has a SPOC domain. Similar to Nito, PPS also forms a complex with Sxl protein and its pre-mRNA. In the future it will be crucial to dissect how different protein domains contribute to the function of SPEN family proteins (Yan, 2015).

Then what is the 'main' role of nito? On one hand, the phenotypes in the sex comb, genitalia and germ line appear specific to Sxl and such phenotypes do not depend on the genetic interaction with other genes in the sex determination pathway. On the other hand, nito clearly has other non-sex-specific functions, as revealed by the lethality, rough eye, and wing phenotype observed in both sexes. Because a null allele of nito is associated with zygotic lethality, the RNAi knockdown approach is a powerful method to reveal sex-related phenotypes. Interestingly, the RNAi screen targeting splicing factors did not identify any new additional sex determination genes, indicating that there are a limited number of genes yet to be identified in this pathway. Finally, intriguingly, three recent studies have identified SPEN and Rbm15 (the mouse and human ortholog of Nito) as factors interacting with Xist, the long noncoding RNA that is essential for dosage compensation in mammals. Clearly, future experiments such as RNA-seq will be necessary to elucidate the mechanism and logic of Nito-mediated signaling events (Yan, 2015).

Rbm15, also known as OTT, was originally identified from infants with acute megakaryoblastic leukemia (AMKL). The t(1, 22) chromosomal translocation results in fusion of RBM15 and MKL1, and the fusion protein is responsible for AMKL development as shown in a mouse model. In addition to this chromosome translocation, recent cancer genome sequencing projects have found that RBM15 and SPEN (also known as SHARP) are mutated in many different types of cancers, such as adenoid cystic carcinomas and bladder cancers. Given that SPEN family proteins are frequently mutated or deleted in cancers, they have been proposed to act as potential tumor suppressors. Studies of Spen and Nito in Drosophila will provide mechanistic insights to understanding of this important family of proteins (Yan, 2015).

Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito

The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. This study shows that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and several phosphorylation sites of Nito were mapped. Furthermore, it was possible to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase (Dewald, 2014).

Spenito and Split ends act redundantly to promote Wingless signaling

Wingless (Wg)/Wnt signaling directs a variety of cellular processes during animal development by promoting the association of Armadillo/β-catenin with TCFs on Wg-regulated enhancers (WREs). Split ends (Spen), a nuclear protein containing RNA recognition motifs (RRMs) and a SPOC domain, is required for optimal Wg signaling in several fly tissues. Spenito (Nito), the only other fly protein containing RRMs and a SPOC domain, acts together with Spen to positively regulate Wg signaling. The partial defect in Wg signaling observed with spen RNAi is enhanced by simultaneous knockdown of nito while it is rescued by expression of nito in wing imaginal discs. In cell culture, depletion of both factors causes a greater defect in the activation of several Wg targets than RNAi of either spen or nito alone. These nuclear proteins are not required for Armadillo stabilization or the recruitment of TCF and Armadillo to a WRE. Loss of Wg target gene activation in cells depleted for spen and nito was not dependent on the transcriptional repressor Yan or Suppressor of Hairless, two previously identified targets of Spen. It is proposed that Spen and Nito act redundantly downstream of TCF/Armadillo to activate many Wg transcriptional targets (Chang, 2008).

Although Drosophila, Spen and Nito are the only two proteins containing both RRMs and a SPOC domain, the sequence similarity of these domains is low and they are unrelated outside of these motifs. Spen and Nito show higher conservation to their respective orthologs in humans than to each other, suggesting that they evolved functional specificity after a duplication event (Jemc, 2006). Consistent with this, Spen and Nito have been shown to act antagonistically in the developing fly eye. Overexpression of nito disrupts eye development, and this phenotype is enhanced by a reduction in spen gene activity. Conversely, a small eye phenotype caused by overexpression of spen^DN is enhanced with nito overexpression and suppressed with nito RNAi (Jemc, 2006). Since Spen has been shown to regulate Notch and EGFR signaling pathways in the eye, it may be that Spen and Nito have opposing functions in these pathways (Chang, 2008).

In this study strong evidence is provided for functional redundancy between spen and nito in the context of Wg signaling. Single and double RNAi analysis indicated that both genes positively regulate the pathway in the fly eye, the wing imaginal disc and in Kc cells. Expression of nito can rescue the spen RNAi phenotype in the wing disc, strongly suggesting that Nito and Spen perform a similar biochemical function in the Wg pathway. There is a report concluding that loss ofspen has no role in Wg signaling in the developing wing. While the discrepancy may be due to the use of different Wg signaling readouts, it is likely that redundancy with nito can explain the negative results obtained with spen mutant clones in that study (Chang, 2008).

In humans, there is evidence that the spen and nito homologs possess both similar and distinct functional activities in the Notch pathway. SHARP can repress Notch signaling through interaction with RBP-Jκ, acting as a transcriptional corepressor. OTT1 can also bind to RBP-Jκ, but this interaction can lead to either repression or activation of a Notch/RBP-Jκ reporter gene, depending on the cell line used (Ma, 2007). Taken together with these data and those of Jemc (2006), the data suggest that Spen and Nito share some biochemical activities but also have distinct, antagonistic properties, depending on the molecular context (Chang, 2008).

In addition to acting redundantly in Wg signaling, it was found that spen and nito are required for the activity of pro-apoptotic factors Hid and Rpr in the fly eye. The fact that the suppression is greatest when both spen and nito are inhibited suggests that they act redundantly, though this requires further study and the molecular relationship between Spen, Nito and apoptosis is not clear (Chang, 2008).

Previous work has demonstrate that spen and nito are required for Arm*, a constitutively active form of Arm, to activate Wg signaling, suggesting that they act downstream of Arm stabilization. In Kc cells, spen, nito depletion did not effect the formation of a TCF-Arm complex on the nkd intronic WRE, even though they are required for activation of nkd expression by Wg signaling. Taken together, these data indicate that Spen and Nito act downstream or in parallel of TCF and Arm to promote transcriptional activation of Wg targets (Chang, 2008).

These results are consistent with those recently reported for SHARP in human cells (Feng, 2007). SHARP was required for maximal activation of Wnt transcriptional targets and reporter constructs, acting downstream of β-catenin stabilization. SHARP could potentiate the ability of Lef1 to activate transcription, independently of Lef1's ability to bind to β-catenin (Feng, 2007). Whether there is a direct biochemical interaction between SHARP and Lef1 is not known (Chang, 2008).

Interestingly, SHARP expression was elevated in several types of carcinomas with constitutively active Wnt/β-catenin signaling, suggesting that it is part of a positive feedback loop regulating the pathway (Feng, 2007). This circuit does not appear to be present in flies, where spen and nito are ubiquitously expressed (Chang, 2008).

Consistent with a role in transcription, Spen and Nito contain domains that could be involved in DNA binding. The murine Spen counterpart Mint has been shown to bind to a G/T-rich element in the FGF-responsive minimal enhancer of the OC promoter via its RRMs. While the recognition site is not well defined, it is interesting to note that there are several G/T-rich regions near some of the functional TCF sites in nkd intronic WRE. Whether the RRMs of Nito or Spen can recognize these sequences is currently being explored (Chang, 2008).

While Spen and Nito may play a direct positive role in the activation of transcriptional targets by Wg signaling, it is also possible that the functional requirement is indirect. The fact that loss of spen elevates Notch signaling in the fly eye fits with the vertebrate data showing that SHARP, Mint and OTT1 can associate with RBP-Jκ and inhibit its ability to activate Notch target genes. However, RNAi inhibition of the fly RBP-Jk homolog Su(H) did not restore activation of Wg targets to spen, nito-depleted cells. This suggests that activation of Notch signaling is not the mechanism by which Spen and Nito promote Wg signaling (Chang, 2008).

Upstream of Su(H), Notch signaling has been reported to repress Wg signaling in flies and fly cell culture. However, this cross-talk has been shown to occur at the level of Arm stabilization. Since Spen and Nito do not effect Arm protein levels, this mechanism appears not be involved in the promotion of Wg signaling by Spen and Nito (Chang, 2008).

Spen is also required to downregulate protein levels of the Ets-domain transcriptional repressor Yan in the eye and wing imaginal discs. Could an increase in Yan levels explain the block in Wg signaling observed when spen and nito are depleted? This does not appear to be the case, since inhibition of yan does not reverse the spen, nito requirement for Wg signaling in Kc cells. In addition, overexpression of Yan in the wing imaginal disc does not affect the expression of the Wg target Sens, which is highly dependent on spen and nito (Chang, 2008).

In contrast to Su(H) and yan, depletion of gro does reverse the spen, nito defect in Wg activation of nkd. Gro is a transcriptional corepressor that is thought to bind to TCF in the absence of Wg signaling and is known to be important in silencing nkd expression. In vitro, binding of β-catenin and TLE1 or TLE2 (vertebrate homologs of Gro) to TCF is mutually exclusive. β-Catenin and TLE1 binding to WREs in cells is also exclusive. This suggests a model where Gro displacement by Arm is defective in spen, nito-depleted cells, leading to reduced activation of nkd. However, it was found that Arm recruitment to the nkd WRE is not affected by spen, nito knockdown. In addition to this discrepancy, Gro has no apparent role in Spen, Nito regulation of CG6234 by the Wg pathway. This suggests that Gro displacement is not the major mechanism by which Spen/Nito function in Wg signaling (Chang, 2008).

One interesting aspect of Spen–Nito regulation of Wg signaling is that all targets of the pathway do not appear to require these proteins for activation. Originally it was shown that Spen is required for Wg function in several imaginal discs, but not in embryos. This could be explained by redundancy with Nito, but ubiquitous knockdown of both genes throughout the embryonic epidermis caused no detectable defect in Wg signaling. In the wing imaginal discs, spen and nito clearly are required for Sens expression, but two other Wg targets, Distal-less and fz3 are expressed normally under the RNAi conditions used. In Kc cells, several Wg targets required spen and nito for activation, but hth did not. These results have to be interpreted cautiously, because residual spen and/or nito activity may be sufficient for regulation of some Wg targets. However, the data do support a model where Spen and Nito regulate Wg-mediated transcriptional activation in a gene-specific manner (Chang, 2008).

In mice, disruption of Mint or OTT1 results in early embryonic lethality. It is interesting to note that a conditional deletion of OTT1 in the hematopoietic compartment caused a defect in pro/pre-B cell differentiation. Loss of Lef1 results in poor survival/growth of pro-B cells. While the phenotypes are not identical, this could be due to redundancy between Lef1 and other TCFs or between Mint and OTT1. The existence of floxed alleles of Mint and OTT1 should allow the relationship between these factors and the Wnt/β-catenin pathway to be more fully explored in the mouse (Chang, 2008).

Characterization of the split ends-like gene spenito reveals functional antagonism between SPOC family members during Drosophila eye development

The novel family of SPOC domain proteins is comprised of broadly conserved nuclear factors that fall into two subclasses, termed large and small, based on protein size. Members of the large subgroup, which includes Drosophila SPEN and human SHARP, have been characterized as transcriptional corepressors acting downstream of a variety of essential cell signaling pathways, while those of the small subclass have remained largely unstudied. Since SPEN has been implicated in Drosophila eye development, and the small SPOC protein (Spenito) Nito is also expressed in the developing eye, this context was used to perform a structure/function analysis of Nito and to examine the relationship between the two SPOC family subclasses. The results demonstrate that the phenotypes obtained from overexpressing Nito share striking similarity to those associated with loss of spen. Dosage sensitive genetic interactions further support a model of functional antagonism between Nito and SPEN during Drosophila eye development. These results suggest that large and small SPOC family proteins may have opposing functions in certain developmental contexts (Jemc, 2006).

spen encodes the founding member of a family of proteins characterized by three N-terminal RNA recognition motifs (RRMs) and a novel C-terminal domain, called the SPEN Paralog Ortholog Conserved domain or SPOC domain. SPEN orthologues have been identified in worms, flies, mosquito, mouse, human, and other vertebrates, and more recent studies have identified proteins in plants and yeast carrying the SPOC domain in conjunction with other functional motifs. The RRMs suggest a role for SPOC family proteins in RNA or DNA binding and in the case of SPEN are necessary for nuclear localization, while the SPOC domain of SPEN and its human and mouse orthologs SHARP (SMRT/HDAC1 Associated Repressor Protein) and MINT (Msx2-interacting nuclear target protein) has been implicated in transcriptional regulation and repression (Kuroda, 2003; Oswald, 2002; Shi, 2001; Yang, 2005). SPOC family proteins can be further divided into two subclasses based on their size. In contrast to large SPOC family proteins almost nothing is known about the functions of small SPOC proteins. Thus far, only the human small SPOC family member One Twenty Two (OTT)/ RNA-Binding Motif protein-15 (RBM15) has been studied. Specifically, chromosomal translocations identified in cases of acute megakaryocytic leukemia revealed a fusion with MAL (Megakaryocytic Acute Leukemia)/MKL1 (Megakaryoblastic Leukemia-1) that results in a chimeric protein that includes almost the entire coding region of both genes, with 4 RBM15/OTT at the N-terminus and MAL/MKL1 at the C-terminus (Ma, 2001; Mercher, 2001). Recent evidence suggests that the RBM15-MKL1 fusion may contribute to leukomogenesis through an increased ability to activate serum response factor (SRF) target genes (Jemc, 2006 and references therein).

Sequence conservation defines two distinct SPOC family subclasses: SPOC family proteins fall into two apparent subclasses based on their size. To determine whether such a distinction might be functionally significant, sequence alignments of the conserved Cterminal SPOC motif were performed to compare the level of sequence conservation in the SPOC family in general and subclass members in particular. Analysis revealed only 27% identity and 50% overall similarity between the SPOC domains of SPEN and Nito, the Drosophila representatives of the large and small subfamilies, respectively; however, upon comparison of the SPOC domains of these proteins with those of their respective subclass family members, a higher level of conservation was revealed. Drosophila SPEN and human SHARP exhibit 58% sequence identity and 79% overall sequence similarity, while Drosophila Nito and human RBM15/OTT share 47% sequence identity and 62% overall sequence similarity. Comparable results were obtained by comparing the RRM motifs. These results reveal a higher level of sequence conservation within SPOC family subclasses relative to the family in general, raising the possibility that subclasses may have adopted divergent functions (Jemc, 2006).

Overexpression of nito perturbs adult eye morphology: To better understand the relationship between large and small SPOC proteins, it was determined if spen and nito function synergistically or antagonistically in vivo. Because the large SPOC family member spen is required for Drosophila eye development and the fly eye provides a uniquely powerful system in which to explore functional relationships between signaling molecules, this analyses focused on the eye. RT-PCR confirmed that nito, like spen, is expressed in the developing eye disc (Jemc, 2006).

Because no nito mutants are currently available, an in vivo structure-function analysis was undertaken to investigate nito function during eye development. While the phenotypes resulting from overexpression of a gene must be interpreted with caution, such overexpression models frequently result in sensitized genetic systems that can provide powerful tools for investigating in vivo relationships between signaling molecules. Myc-tagged full-length Nito (Nito-FL), Nito lacking the N-terminus (NitoDN; an exogenous nuclear localization sequence was added to ensure proper nuclear targeting) and Nito lacking the C-terminus (NitoDC) were cloned downstream of a UAS promoter and the transgenes were expressed in flies using eye specific GAL4 drivers. Three different sevenless-Gal4 (sev-Gal4) drivers, which promote expression in photoreceptors R1, R3, R4, R6, R7, the cone cells and the 'mystery' cells, which are poorly understood interommatidial cells that are never recruited to the ommatidia and ultimately apoptose, were utilized in this study: sev^strong couples the sev enhancer to the hsp70 promoter, resulting in the highest levels of expression; sevmedium contains both the sev enhancer and sev promoter, and expresses at an intermediate level; sev^weak contains the same regulator sequences as ^sevmedium, but expresses at lower levels, presumably as a consequence of position effect of the transgene. To avoid unnecessary confusion, these will be referred to collectively as sev-Gal4 (Jemc, 2006).

Sev-Gal4 driven overexpression of Nito-FL and NitoDC yielded dosage dependent adult rough eye phenotypes, while overexpression of NitoDN was indistinguishable from wild type. Western blots confirmed the expression of all transgenes, and immunohistochemistry showed nuclear localization of Nito in all cases, indicating that the lack of a NitoDN phenotype is not due to the absence or mislocalization of protein. While overexpression of Nito-FL and NitoDC both perturb eye development, the resulting phenotypes are distinct. This data is not unexpected given previous results for the large SPOC family protein, SPEN, in which overexpression of SPENDC functions as a dominant negative with respect to spen (Jemc, 2006).

It was therefore speculated that NitoDC functions analogously as a dominant negative relative to nito whereas Nito-FL expression simply augments the pool of full-length Nito. Specifically, it was observed that overexpression of Nito-FL results in roughening of the posterior part of the eye and an overall decrease in eye size, whereas overexpression of NitoDC more uniformly perturbs the external morphology of the eye (Jemc, 2006).

In order to distinguish the Nito-FL and NitoDC rough eye phenotypes at the cellular level, adult eyes were sectioned and examined for defects. In wildtype ommatidia, photoreceptors are arranged in a trapezoidal array with seven of the eight photoreceptors visible in one plane of view. The regular trapezoidal arrangement of photoreceptors is disturbed in both overexpression systems. When Nito-FL is overexpressed, a decrease in the number of photoreceptors per ommatidia, elongated rhabdomeres, as well as a general disorganization of the ommatidia are seen. These observations suggest that the rough eye phenotype is due to a loss of photoreceptors and possible defects in the accessory cells, which normally provide support for the rhabdomeres in the ommatidia. This phenotype is strikingly reminiscent of that seen in sections of spen mutant eye clones, raising the possibility that overexpressed nito may function antagonistically with respect to spen in the developing eye (Jemc, 2006).

Eyes overexpressing NitoDC also appear disorganized compared to wildtype, although in contrast to Nito-FL ommatidia, photoreceptor number is not strongly affected. Rather, the most prevalent defect appears to be ommatidial fusions suggesting that cone and pigment cells, rather than photoreceptors are most affected. Given that the Gal4 driver used for these experiments is expressed primarily in a subset of photoreceptors, the cone cells and interommatidial mystery cells, the accessory cell defects observed upon nito overexpression may be due in part to indirect effects on pigment cells. Thus, Nito-FL ommatidia have defects in photoreceptor number and ommatidial morphology, while NitoDC ommatidia have defects in accessory cells required for the spacing of ommatidia (Jemc, 2006).

To further investigate the defects caused by overexpressing nito, the effects of increasing nito expression in early eye development were examined. First, recruitment of the photoreceptor neurons into ommatidia was examined by looking at expression of the panneural marker ELAV in the larval precursor to the eye, the eye imaginal disc. Consistent with the differences observed in the adult phenotypes, the larval phenotypes associated with sev-Gal4 driven expression of Nito-FL and NitoDC are also distinct. In eye discs overexpressing Nito-FL, initial recruitment of photoreceptors appears normal. However, approximately seven rows posterior to the furrow, there is a decrease in the number of photoreceptors per ommatidia. Thus while Nito-FL expression does not perturb initial photoreceptor recruitment, subsequent development and/or survival are compromised, resulting in the reduced number of photoreceptors observed in the adult eye. The loss of photoreceptors upon overexpression of Nito-FL is also similar to spen mutant clones, which have reduced numbers of photoreceptors in mutant ommatidia in the developing imaginal disc, consistent with the observations made in adult eye sections. In contrast to Nito-FL and spen mutant clones, and consistent with the ommatidial fusions observed in adult eye sections, overexpression of NitoDC causes loss of spacing between ommatidia in the larval eye disc, while recruitment of photoreceptors is not affected (Jemc, 2006).

To examine the possibility that the phenotypes associated with overexpression of Nito-FL and NitoDC were due primarily to cell death, eye discs were stained with the apoptotic marker acridine orange. In the wildtype eye disc very little cell death is observed. In Nito-FL eye discs, a stripe of cell death occurs in the posterior part of the differentiating eye disc, consistent with the loss of photoreceptors observed in the ELAV-probed eye disc and similar to the elevated cell death phenotype observed in spen mutant clones. However coexpression of the apoptotic inhibitor p35 or introduction of the H99 Deficiency that removes the proapoptotic genes hid, reaper and grim, did not suppress the Nito-FL rough eye phenotypes, suggesting that increased apoptotic cell death is unlikely to be the primary factor contributing to the Nito-FL associated eye defects. In discs overexpressing NitoDC, increased cell death is observed more anteriorly relative to that for Nito-FL, consistent with the ommatidial spacing defects observed in the ELAV-probed disc (Jemc, 2006).

The potential for functional antagonism between SPEN and Nito was suggested by the similarity of phenotypes observed in adult eye sections overexpressing Nito-FL and in spen mutant eye clones. To further investigate this potential antagonism, a series of dose-sensitive genetic interactions between spen and nito was performed (Jemc, 2006).

Initially, the effects of reducing spen levels in the Nito-FL overexpression background was examined. If Nito-FL antagonizes SPEN function, as suggested by the phenotypic analysis, further reducing spen should exacerbate the Nito-FL overexpression phenotype. An important requirement for such an experiment is the need for dose-sensitive Nito-FL phenotypes. Two observations suggest Nito-FL provides a dose-sensitive phenotype ideal for studying genetic interactions: (1) expression of independent transgenic lines with the same sev-Gal4 driver results in a range of phenotypes, and (2) expression of a given Nito-FL transgene with sev^weak results in a mild rough eye phenotype whereas expression of the same line at a higher level produces a more severe phenotype. Consistent with the hypothesis of an antagonistic relationship between spen and nito, it was found that heterozygosity for a null spen allele enhanced the rough eye phenotype associated with Nito-FL expression, as demonstrated by an increased number of ommatidia lacking photoreceptors. Next, the consequences of increasing or decreasing nito levels in the background of a dominant negative spen transgene (spenDN), which encodes the C-terminal 936 amino acids of spen), and also produces dose sensitive phenotypes, that were examined. Because both transgenes are capable of perturbing eye development on their own, in order to distinguish between additive and synergistic interactions a Nito-FL transgenic line was used that when expressed with sev^weak exhibits only very mild perturbations of the adult eye (Jemc, 2006).

As expected, given the Nito structure-function analysis, Nito-FL causes an enhancement of the spenDN rough eye phenotype, an increase in necroses in the eye and a complete loss of organization. Thus, overexpression of nito and overexpression of spenDN appear to act in the same direction, suggesting opposing functions for Nito and Spen. Since loss-of-function mutations in nito have not been isolated, a nito transgenic dsRNA construct was generated to investigate the consequences of reducing endogenous nito expression levels with respect to spen function. RT-PCR from Drosophila eye discs confirmed that this construct mediates partial knockdown of nito expression. In vivo, while dsRNA-mediated knockdown of nito expression does not perturb eye morphology on its own, nito-RNAi partially rescues the rough eye phenotype resulting from overexpression of spenDN, again suggesting antagonism between nito and spen. Eye sections show fewer missing ommatidia in nito-RNAi, spenDN adult eyes relative to those overexpressing spen DN alone, as well as fewer missing photoreceptors in ommatidia lacking the full complement of photoreceptors and more normal rhabdomere morphology. Together, these dose-sensitive genetic interactions argue for mutual antagonism between the large SPOC family member spen and the small SPOC family representative nito during Drosophila eye development (Jemc, 2006).

It remains to be determined if the antagonistic relationship between nito and spen is maintained in developmental contexts outside of the eye. Previous work examining the role of SPEN in Wingless signaling suggested the presence of a redundant partner for SPEN, for which Nito would be a good candidate, given their sequence conservation. In situ hybridization for nito and spen suggests they are also both ubiquitously expressed throughout embryonic development, and considering the broad range of embryonic phenotypes attributed to spen mutants, exploration of context specific interactions between spen and nito in the embryo will likely improve understanding of the relationships between these two related proteins. It is predicted that certain developmental events will require synergism between nito and spen, whereas others, as was demonstrate in the eye, will require antagonism (Jemc, 2006).

At the cellular level, spen is implicated as a positive component of Wingless and RTK/RAS signaling, and large SPOC family proteins SHARP and MINT are implicated as negative regulators of Notch signaling (Kuroda, 2003; Oswald, 2002). Given the ability of nito to antagonize spen function in the developing eye, it seems reasonable to speculate that Nito also acts as a downstream regulator/effector of some or all of these pathways. Furthermore, the antagonism between nito and spen may provide a mechanism for differential regulation of output from these pathways. Mechanistically, how might one envision the mutual antagonism between Spen and Nito? Large SPOC proteins have been previously shown to serve as transcriptional corepressors. Thus one attractive possibility is that small SPOC proteins might serve as transcriptional activators. In this model, by virtue of their conserved RRM and SPOC motifs, small and large SPOC proteins might compete for access to common binding partners. The resulting complexes, depending on whether they contain Spen or Nito, would then either repress or activate transcription. In a slight variation of the model, one could propose that SPOC proteins might be able to either repress or activate transcription, and so depending on context, would either act synergistically or antagonistically. Unfortunately, Drosophila cultured cells do not provide an appropriate environment in which to assay the activity of SPOC proteins so it was not possiable to test this model with respect to Spen and Nito. However, using mammalian COS cells, it was observed that while the SPOC motif of SHARP represses transcription, the SPOC motif of RBM15, the human Nito ortholog, strongly activates transcription. Thus, perhaps the antagonistic relationship between Spen and Nito that is reported in this study in the context of Drosophila eye development reflects a conserved antagonistic relationship between large and small SPOC proteins that is manifested at the level of transcriptional output (Jemc, 2006).

In conclusion, an antagonistic relationship has been demonstrated between the large and small SPOC family proteins in the developmental context of the Drosophila eye. The finding that SPOC family proteins function as downstream effectors of a variety of signaling pathways suggests they may act to fine-tune transcriptional output downstream of these cascades. Thus, it will be extremely interesting to determine whether the antagonistic relationship have observed between Nito and Spen in the eye is a general property of small and large SPOC proteins, or if it is unique to Drosophila eye development. Determination of transcriptional targets and cofactors will be required to understand how SPOC family proteins function to regulate and integrate information from these signaling pathways (Jemc, 2006).

Search PubMed for articles about Drosophila Spenito

Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. and Rottman, F. M. (1997). Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3(11): 1233-1247. PubMed ID: 9409616

Chang, J. L., Lin, H. V., Blauwkamp, T. A. and Cadigan, K. M. (2008). Spenito and Split ends act redundantly to promote Wingless signaling. Dev. Biol. 314(1): 100-11. PubMed Citation: 18174108

Dewald, D. N., Steinmetz, E. L. and Walldorf, U. (2014). Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito. Insect Mol Biol. PubMed ID: 25040100

Feng, Y.,, (2007). Drosophila split ends homologue SHARP functions as a positive regulator of Wnt/β-Catenin/T-cell factor signaling in neoplastic transformation. Cancer Res. 67: 485-491. PubMed Citation: 17234755

Gu, T., Zhao, T., Kohli, U. and Hewes, R. S. (2017). The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila. Dev Biol 431(2):226-238. PubMed ID: 28916169

Hazegh, K. E., Nemkov, T., D'Alessandro, A., Diller, J. D., Monks, J., McManaman, J. L., Jones, K. L., Hansen, K. C. and Reis, T. (2017). An autonomous metabolic role for Spen. PLoS Genet 13(6): e1006859. PubMed ID: 28640815

Jemc, J. and Rebay, I. (2006). Characterization of the split ends-like gene spenito reveals functional antagonism between SPOC family members during Drosophila eye development. Genetics 173(1): 279-86. 16547102

Knuckles, P., Lence, T., Haussmann, I. U., Jacob, D., Kreim, N., Carl, S. H., Masiello, I., Hares, T., Villasenor, R., Hess, D., Andrade-Navarro, M. A., Biggiogera, M., Helm, M., Soller, M., Buhler, M. and Roignant, J. Y. (2018). c3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m(6)A machinery component Wtap/Fl(2)d. Genes Dev. PubMed ID: 29535189

Lence, T., Akhtar, J., Bayer, M., Schmid, K., Spindler, L., Ho, C. H., Kreim, N., Andrade-Navarro, M. A., Poeck, B., Helm, M. and Roignant, J. Y. (2016). m6A modulates neuronal functions and sex determination in Drosophila. Nature 540(7632): 242-247. PubMed ID: 27919077

Liu, J., Yue, Y., Han, D., Wang, X., Fu, Y., Zhang, L., Jia, G., Yu, M., Lu, Z., Deng, X., Dai, Q., Chen, W. and He, C. (2014). A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10(2): 93-95. PubMed ID: 24316715

Yan, D. and Perrimon, N. (2015). spenito is required for sex determination in Drosophila melanogaster. Proc Natl Acad Sci 112(37): 11606-11. PubMed ID: 26324914

date revised: 5 August 2023

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