InteractiveFly: GeneBrief

P-element somatic inhibitor: Biological Overview | References

The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, this study targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits that were identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, it is concluded that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern (Foley, 2019).

Increasing evidence indicates that post-transcriptional mechanisms controlling gene expression are also critical for the proper function of circadian clocks in many organisms. In Drosophila, the post-transcriptional regulation of per mRNA has been best studied. per mRNA stability changes as a function of time. In addition, per contains an intron in its 3'UTR (dmpi8) that is alternatively spliced depending on temperature and lighting conditions. On cold days, the spliced variant is favored, causing an advance in the accumulation of per transcript levels as well as an advance of the evening activity peak. This behavioral shift means that the fly is more active during the day when the temperature would be most tolerable in their natural environment. The temperature sensitivity of dmpi8 is due to the presence of weak non-canonical splice sites. However, the efficiency of the underlying baseline splicing is affected by four single nucleotide polymorphisms (SNPs) in the per 3'UTR that vary in natural populations and form two distinct haplotypes. Also, while this splicing is temperature-sensitive in two Drosophila species that followed human migration, two species that remained in Africa lack temperature sensitivity of dmpi8 splicing. Furthermore, Zhang (2018) recently demonstrated that the the trans-acting splicing factor B52 enhances dmpi8 splicing efficiency, and this effect is stronger with one of the two haplotypes. per is also regulated post-transcriptionally by the TWENTYFOUR-ATAXIN2 translational activation complex (see Lim, 2013). This complex works by binding to per mRNA as well as the cap-binding complex and poly-A binding protein. This may enable more efficient translation by promoting circularization of the transcript. Interestingly, this mechanism appears to be required only in the circadian pacemaker neurons. Non-canonical translation initiation has also been implicated in the control of PER translation. Regulation of PER protein translation has also been studied in mammals, with RBM4 being a critical regulator of mPER1 expression. In flies however, the homolog of RBM4, LARK, regulates the translation of DBT, a PER kinase. miRNAs have emerged as important critical regulators of circadian rhythms in Drosophila and mammals, affecting the circadian pacemaker itself, as well as input and output pathways controlling rhythmic behavioral and physiological processes (Foley, 2019).

RNA-associated proteins (RAPs) include proteins that either bind directly or indirectly to RNAs. They mediate post-transcriptional regulation at every level. Many of these regulated events - including alternative splicing, splicing efficiency, mRNA stability, and translation - have been shown to function in molecular clocks. Thus, to obtain a broad view of the Drosophila circadian RAP landscape and its mechanism of action, an RNAi screen was performed targeting 364 of these proteins. This led to the discovery of a role for the splicing factor P-element somatic inhibitor (PSI) in regulating the pace of the molecular clock through alternative splicing of tim (Foley, 2019).

The results identify a novel post-transcriptional regulator of the circadian clock: PSI. PSI is required for the proper pace of both brain and body clock, and for proper phase-relationship with ambient temperature cycles. When Psi is downregulated, the circadian pacemaker speeds up and behavior phase under temperature cycles is advanced by 3 hr, and these phenotypes appear to be predominantly caused by an abnormal tim splicing pattern. Indeed, the circadian period and behavior phase of flies that can only produce functional TIM protein from a transgene missing most introns is insensitive to Psi downregulation. It is noted however that cwo's splicing pattern is also affected by Psi downregulation, and sgg splicing pattern was not studied, although it might also be controlled by PSI. It is therefore not possible to exclude a small contribution of non-tim splicing events to PSI downregulation phenotypes, or that in specific tissues these other splicing events play a greater role than in the brain (Foley, 2019).

Interestingly, Psi downregulation results in an increase in intron inclusion events that are favored under cold conditions (tim-sc and tim-cold), while an intron inclusion event favored under warm conditions is decreased (tim-M). However, the ability of tim splicing to respond to temperature changes is not abolished when Psi is downregulated. This could imply that an as yet unknown factor specifically promotes or represses tim splicing events in a temperature-dependent manner. Another possibility is that the strength of splice sites or tim's pre-mRNA structure impacts splicing efficiency in a temperature-dependent manner. For example, suboptimal per splicing signals explain the lower efficiency of per's most 3' splicing event at warm temperature (Foley, 2019).

How would the patterns of tim splicing affect the pace of the circadian clock, or advance the phase of circadian behavior under temperature cycles? In all splicing events that were studied, intron retention results in a truncated TIM protein. It is therefore possible that the balance of full length and truncated TIM proteins, which may function as endogenous dominant-negatives, determines circadian period. For example, truncated TIM might be less efficient at protecting PER from degradation, thus accelerating the pacemaker, or affecting its phase. Consistent with this idea, overexpression of the shorter cold-favored tim isoform (tim-sc) shortens period (Martin Anduaga, 2019). Strikingly, Psi downregulation increases this isoform's levels and also results in a short phenotype. Shakhmantsir (2018) also proposed that production of tim-M transcripts (called tim-tiny in their study) delays the rate of TIM accumulation. Such a mechanism could also contribute to the short period observed when Psi is downregulated, since this reduces tim-M levels, which may accelerate TIM accumulation. Another interesting question is how PSI differentially affects specific splice isoforms of tim. One possibility is that the execution of a specific tim splicing event negatively influences the probability of the occurrence of other splicing events. For example, PSI could downregulate tim-sc and tim-cold by enhancing splicing and removal of the introns whose retention is necessary for production of these isoforms. This could indirectly reduce splicing of the intron that is retained in the warm tim-M isoform and result in tim-M upregulation. Conversely, PSI could directly promote tim-M intron retention and indirectly downregulate production of tim-sc and tim-cold (Foley, 2019).

Other splicing factors have been shown to be involved in the control of circadian rhythms in Drosophila. SRm160 contributes to the amplitude of circadian rhythms by promoting per expression, while B52/SMp55 and PRMT5 regulate per's most 3' splicing, which is temperature sensitive. Loss of PRMT5 results in essentially arrhythmic behavior, but this is unlikely to be explained by its effect on per's thermosensitive splicing. B52/SMp55 knockdown flies show a reduced siesta, which is controlled by the same per splicing (Zhang, 2018). With the identification of Psi, this study has uncover a key regulator of tim alternative splicing pattern and shows that this pattern determines circadian period length, while per alternative splicing regulates the timing and amplitude of the daytime siesta. Interestingly, a recent study identified PRP4 kinase and other members of tri-snRNP complexes as regulators of circadian rhythms (Shakhmantsir, 2018). Downregulation of prp4 caused excessive retention of the tim-M intron. PSI and PRP4 might thus have complementary functions in tim mRNA splicing regulation, working together to maintain the proper balance of tim isoform expression (Foley, 2019).

An unexpected finding is the role played by both PDF neurons and other circadian neurons in the short period phenotype observed with circadian locomotor rhythms when Psi was knocked-down. Indeed, it is quite clear from multiple studies that under constant darkness, the PDF-positive sLNvs dictate the pace of circadian behavior. Why, in the case of Psi downregulation, do PDF negative neurons also play a role in period determination? The explanation might be that PSI alters the hierarchy between circadian neurons, promoting the role of PDF negative neurons. This could be achieved by weakening PDF/PDFR signaling, for example (Foley, 2019).

While this study focused on PSI, several other interesting candidates were identified in the screen. The presence of a large number of splicing factors is noted. This adds to the emerging notion that alternative splicing plays a critical role in the control of circadian rhythms. Several per splicing regulators have been mentioned that can impact circadian behavior. In addition, a recent study demonstrated that specific classes of circadian neurons express specific alternative splicing variants, and rhythmic alternative splicing is widespread in these neurons. Interestingly, in this study, the splicing regulator barc, which was identified in the screen and which has been shown to causes intron retention in specific mRNAs, was found to be rhythmically expressed in LNds. Moreover, in mammals, alternative splicing appears to be very sensitive to temperature, and could explain how body temperature rhythms synchronize peripheral clocks. Another intriguing candidate is cg42458, which was found to be enriched in circadian neurons (LNvs and Dorsal Neurons 1). In addition to emphasizing the role of splicing, the screen suggests that regulation of polyA tail length is important for circadian rhythmicity, since several members of the CCR4-NOT complex and deadenylation-dependent decapping enzymes were identified. Future work will be required to determine whether these factors directly target mRNAs encoding for core clock components, or whether their effect on circadian period is indirect. Interestingly, the POP2 deadenylase, which is part of the CCR4-NOT complex, was recently shown to regulate tim mRNA levels post-transcriptionally. It should be noted that while the screen targeted 364 proteins binding or associated with RNA, it did not include all of them. For example, LSM12, which was recently shown to be a part of the ATXN2/TYF complex, was not included in the screen because it had not been annotated as a potential RAP when the screen was initiated (Foley, 2019).

In summary, this work provides an important resource for identifying RNA associated proteins regulating circadian rhythms in Drosophila. It identifies PSI is an important regulator of circadian period and circadian phase in response to thermal cycles, and points at additional candidates and processes that determine the periodicity of circadian rhythms (Foley, 2019).

Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila

This study reports novel tumor suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, these data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth (Zaytseva, 2020).

Tightly coordinated regulation of cell and tissue growth is essential for animal development. Decreased growth leads to small organs and diminished body size, whereas heightened proliferative growth is associated with genomic instability and cancer. The MYC transcription factor and growth regulator has been studied extensively since its identification as an oncogene in the early 1980s, when MYC overexpression caused by chromosomal translocation was found to drive malignant transformation in Burkitt's lymphoma. Research in subsequent decades implicated increased MYC in progression of most tumours. In normal adult tissues, MYC expression is relatively low and generally restricted to cells with regenerative and proliferative potential. Even small increases in MYC abundance are sufficient to promote proliferative cell growth; thus, understanding the molecular control of MYC expression can provide crucial insight into the mechanisms of MYC dysregulation in cancer (Zaytseva, 2020).

In normal cells, MYC is regulated by signalling inputs from a diverse array of developmental and growth signalling pathways. The many cellular signalling inputs converging on MYC transcription are integrated by FUBP1, a KH domain protein that binds single-stranded DNA and interacts with the general transcription factor complex TFIIH to modulate MYC promoter output. The mammalian FUBP family comprises three proteins (FUBP1-3) that are represented by one orthologue in Drosophila, P-element somatic inhibitor (Psi). Like FUBP1, Psi also interacts with the RNA polymerase II (RNA Pol II) transcriptional machinery, particularly the transcriptional Mediator (MED) complex, to pattern Myc transcription and cell and tissue growth in the Drosophila wing epithelium (Guo, 2016). In addition to roles in transcription, Psi binds RNA via the KH domains and interacts with the spliceosome to regulate mRNA splicing. Although co-immunoprecipitation (co-IP) mass spectrometry detected Psi in complex with the Argonaute protein AGO1, the potential significance of this interaction is unknown (Zaytseva, 2020).

Argonaute proteins comprise the core of the RNA-induced silencing complex (RISC), which uses noncoding RNA as a guide to target mRNAs for post-transcriptional gene silencing. Drosophila AGO2 is best characterised as part of the siRNA-induced silencing complex (siRISC), whereas AGO1 predominantly functions in microRNA-induced silencing complexes (miRISCs) and post-transcriptional mRNA silencing. Of importance to this study, AGO1-mediated mRNA silencing has been implicated in transcript destabilisation and translational repression of Myc in flies and humans. This study reports a novel role for AGO1 as a direct Myc transcriptional repressor and demonstrates that this underlies cell growth inhibition. AGO1 depletion not only increases Myc promoter activity, mRNA and protein abundance, but the elevated Myc expression requires RNA Pol II transcriptional activity. Localisation to the nucleus, together with interaction with transcriptional machinery and significant AGO1 enrichment on the Myc promoter suggests, in addition to the established roles in miRNA silencing in the cytoplasm, AGO1 constrains Myc transcription to control cell and tissue growth during Drosophila development (Zaytseva, 2020).

This study demonstrates a novel role for AGO1 as a growth inhibitor in Drosophila. AGO1 depletion was sufficient to increase Myc (mRNA and protein) to drive ribosome biogenesis, nucleolar expansion and cell growth in a Myc and Psi-dependent manner. The increased Myc promoter activity in AGO1 knockdown wing discs, together with the α-amanitin-dependent increase in Myc pre-mRNA abundance, suggests that AGO1 represses Myc at the level of transcription. In accordance with the observed growth inhibitory capacity of AGO1, the increased Myc mRNA and protein abundance in AGO1 knockdown wings were associated with increased Myc function (i.e. activation of established Myc targets). Interestingly, although Psi co-knockdown only modestly decreased Myc mRNA levels in AGO1-depleted wings, Psi co-depletion strongly reduced expression of Myc targets. This observation suggests that Psi is not only required for Myc transcription (Guo, 2016) but may also be required for activation of Myc growth targets in the context of AGO1 depletion. Thus, future studies are required to determine whether Psi and Myc bind common targets and whether Psi is required for transcriptional activation of Myc target genes (Zaytseva, 2020).

Recent genome-wide functional RNAi screens in Drosophila S2 cells, identifying AGO1 as a modifier of Polycomb foci, suggested extra-miRNA functions for AGO1. PcG mediates epigenetic repression of key developmental genes to control cell fate, and PcG repression is stabilised via aggregation of PcG foci in the nucleus. AGO1 depletion disrupted nuclear organisation and reduced the intensity of Pc foci, suggesting that AGO1 negatively regulates PcG-mediated silencing. The Drosophila PcG complex has been characterised for roles in silencing homeotic genes by binding PcG response elements (PREs), including the Fab-7 PRE-containing regulatory element from the Hox gene, Abdominal-B. Components of the RNAi machinery, including AGO1 and Dicer-2, have been implicated in driving PcG-dependent silencing between remote copies of the Fab-7 element, engineered throughout the genome to monitor long-distance gene contacts. Interactions between Hox genes silenced by PcG proteins were decreased in AGO1 mutants, suggesting that AGO1 regulates nuclear organisation, at least in part, by stabilising PcG protein recruitment to chromatin (Zaytseva, 2020).

Myc transcriptional autorepression, modelled in the Drosophila embryo via overexpression of Myc from an exogenous promoter, leads to repression of the endogenous Myc locus in a Pc-dependent manner (Goodliffe, 2005). This, together with the partial overlap between AGO1 and PcG in wing imaginal disc cells, suggests that Pc mediates transcriptional autorepression of Myc via AGO1. In contrast to the current studies, where AGO1 depletion phenotypes are associated with a moderate (>three- to fivefold) increase in Myc, autoregulation in the embryo was investigated in response to non-physiological increases in Myc (over 100 times endogenous levels) (Goodliffe, 2005). Thus, the current data suggest that AGO1 binds the Myc promoter under normal conditions and is required for repression of endogenous Myc transcription, but whether AGO1 is required for Pc-dependent Myc autorepression requires further investigation. In a similar vein, super-enhancers control human MYC transcription via CTCF in the context of high-MYC cancers (s). Thus, failed Pc-dependent autorepression and/or defective repression of super-enhancers via CTCF could further elevate MYC to promote cancer progression. Given the observed overlap between AGO1 and Pc/CTCF in the Drosophila wing, future studies determining whether AGO1 interacts with Pc and/or CTCF to control autoregulatory feedback on Myc transcription in the context of tumorigenesis will be of great interest (Zaytseva, 2020).

The question remains regarding how AGO1 targets Myc transcription. The physical and genetic interaction between Psi and AGO1, and the observation that AGO1 loss-of-function mutants restore cell and tissue growth in the Psi knockdown wing, suggests that AGO1 inhibits growth that is dependent on this Myc transcriptional regulator. AGO2 has been implicated in insulator-dependent looping interactions defining 3D transcriptional domains (TADs) through association with CTCF binding sites in Drosophila. Although similar roles for AGO1 have not been reported, the cancer-related super-enhancers for the MYC oncogene lie within the 2.8 Mb TAD and control MYC transcription via a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter; that is, in proximity with the FUSE (1.7 kb upstream) bound by FUBP1. Moreover, gene disruption of the enhancer-docking site reduces CTCF binding and super-enhancer interaction, which results in reduced MYC expression and proliferative cell growth (Schuijers, 2018). AGO1 ChIP revealed significant enrichment on the Myc promoter, suggesting that AGO1 probably interacts with Psi and the RNA Pol II machinery to directly regulate Myc transcription. Given the high level of conservation between AGO and CTCF proteins throughout evolution, it is of great interest to determine whether human AGO1 also interacts with FUBP1 to regulate transcription of the MYC oncogene (Zaytseva, 2020).

This study has shown that AGO1 behaves as a growth inhibitor during Drosophila development, through the ability to suppress Myc transcription, ribosome biogenesis and cell growth in the wing disc epithelium. Consistent with AGO1 having tumour suppressor activity, across a wide range of human cancers, large scale genomics data in cBioPortal identified AGO1 as frequently mutated or deleted in a diverse variety of tumours (e.g. reproductive, breast, intestinal, bladder, and skin cancers). Region 1p34-35 of chromosome 1, which includes AGO1, is frequently deleted in Wilms' tumours and neuro-ectodermal tumours. In neuroblastoma cell lines, AGO1 behaves as a tumour suppressor, with overexpression heightening checkpoint sensitivity and reducing cell cycle progression. GEO Profile microarray data inversely correlates AGO1 expression with proliferative index; that is, AGO1 levels are significantly lower in tumorigenic cells than in differentiated cells. In the context of cancer, it is important to determine whether AGO1 loss of function alters MYC-dependent cancer progression and vice versa. As increased abundance of the MYC oncoprotein is associated with the pathogenesis of most human tumours, deciphering such novel mechanisms of MYC repression is fundamental to understanding MYC-dependent cancer initiation and progression (Zaytseva, 2020).

The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila

Fruitless alternative pre-mRNA splicing (AS) isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, this study shows that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ~1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior (Wang, 2016).

How gene regulation modulates neuronal activities leading to cognition and behavior is an important question in biology. Although many behavior-associated genes and neuronal cell types have been identified, a detailed understanding that links the molecular events of gene regulation to specific behaviors is still lacking. Alternative pre-mRNA splicing (AS) is a crucial gene regulatory mechanism that enables a single gene to generate functionally distinct messenger RNA transcripts (mRNAs) and protein products. The nervous system makes extensive use of AS to generate diverse and complex neural mRNA expression patterns that determine numerous neuronal cell types and functions. AS is regulated by the small nuclear ribonucleoprotein complexes (snRNPs) that compose the spliceosome for intron recognition and removal, as well as a large repertoire of non-snRNP RNA-binding proteins that affect decisions on splice site use. This dynamic and complex AS regulatory network modulates diverse neuronal functions, like synaptic transmission and signal processing, hence further impacting higher brain functions, such as cognition and behavioral control (Wang, 2016).

The Drosophila KH-domain RNA binding splicing factor P-element somatic inhibitor (PSI) is best known for regulating tissue-specific AS of the Drosophila P-element transposon transcripts to restrict transposition activity to germ-line tissues. PSI directly interacts with the U1 snRNP through a 70-aa tandem direct repeat domain at the C terminus of the PSI protein (termed the 'AB' domain). Deletion of the AB domain in transgenic flies resulted in male sterility and male courtship defects. U1 snRNP, as an essential component of the spliceosome that binds to 5' splice sites (5'SS), defines exon-intron boundaries, and initiates spliceosome assembly for intron removal. U1 snRNP further affects AS decisions and suppresses pre-mRNA premature cleavage and polyadenylation through binding to pseudo-5'SS (5'SS-like motifs that are not used for splicing) that are abundantly distributed throughout the transcriptome. It remains a mystery how U1 snRNP differentiates the vast number of functional 5'SS and pseudo-5'SS in the transcriptome that leads to functionally distinct AS patterns. In the case of Drosophila P-element transposon AS regulation, the PSI-U1 snRNP interaction enables PSI to modulate the competitive binding of U1 snRNP between the accurate 5'SS in the third intron and an upstream pseudo-5'SS in the transposon pre-mRNA, and thus influence the final AS decision. It is possible that PSI may play a more general role in specifically localizing U1 snRNP to the transcriptome for AS regulation beyond the P-element transposon, and thus exert a more broad influence over fruit fly physiology (Wang, 2016).

AS patterns are often controlled by the interaction of RNA binding proteins (RBPs) with nascent pre-mRNA transcripts. These RNA-protein interactions can determine where the spliceosomal U1 and U2 snRNPs bind to the transcriptome, and thus dictate AS decisions and constitute an important mechanism for gene regulation. RBPs, such as PSI or TIA-1, which directly interact with U1 snRNP, are good candidates for proteins controlling AS patterns in this manner, and changes in these RBP-snRNP associations can have profound phenotypic effects. For example, this study shows that a subtle mutation that abolishes the PSI-U1 snRNP interaction dramatically changed the AS patterns of hundreds neuronal pre-mRNAs and resulted in highly abnormal male courtship behaviors. Given the diverse number of cell types, gene-expression patterns, and the extensive AS that occurs in animal nervous systems, it is anticipated that AS regulation will play critical roles in both the normal physiological or disease states of neurons (Wang, 2016).

The PSI-U1 snRNP interaction may further play crucial roles in other pre-mRNA processing pathways. For example, U1 snRNP was recently ascribed a new function in regulating global mRNA 3' end termination and suppression of premature pre-mRNA cleavage and polyadenylation near the 5' ends of transcripts in humans, mice, and Drosophila through selective binding to 5'SS-like motifs, a process called telescripting. It has remained a mystery how U1 snRNP discriminates the numerous potential 5'SS sites across the transcriptome. PSI may be one example of RBP regulators that alter the binding of U1 snRNP to pre-mRNA sites through direct protein-protein interactions, and thus changing pre-mRNA splicing, polyadenylation, or other pre-mRNA processing patterns (Wang, 2016).

These findings further reveal that even broadly expressed RBPs, such as PSI, can affect gene regulation in restricted subsets of neurons in the Drosophila brain that modulate specific behaviors, such as courtship and mating. The work presented here also provides the first identification of the PSI protein as a transacting RNA splicing factor controlling male-specific fruitless splicing (Wang, 2016).

Taken together, these results link the molecular interaction between PSI and U1 snRNP to specific phenotypic effects on Drosophila courtship behavior through the coordination of an AS program in the brain. These results provide important insights into the mechanisms controlling gene activity in the nervous system, leading to the precise control of complex animal behaviors (Wang, 2016).

Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, this study demonstrates the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and this study demonstrates that Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. The data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, it is predicted that interaction between MED and FBP1 might also have implications for cancer initiation and progression (Guo, 2016).

The capacity to integrate extracellular growth and developmental signals is fundamental to coordinated growth of organs and tissues in multicellular animals. Although the MED complex is required for most (if not all) RNA Pol II dependent transcription, the CDK8 module in particular has been noted for its capacity to sense developmental and environmental cues to activate specific transcriptional networks. In Drosophila, MED responds to specific developmental networks to control patterning of the wing imaginal disc. Consistent with specific roles in regulating growth networks, CDK8 has also been implicated as a negative regulator of tissue growth in mice. The observation that Psi contains potent transcriptional activation capacity suggest Psi helps activate endogenous RNA Pol II activity on dMYC to modulate dMYC transcription and control tissue growth during development. Together with the impaired growth phenotype associated with Psi depletion being suppressed following either co-depletion of subunits from the transcriptionally repressive CDK8/CycC kinase module or overexpression of core subunits, these findings suggests Psi/dMYC-dependent tissue growth depends on MED abundance and activity (Guo, 2016).

Thus, in Drosophila Psi and MED integrate growth signals to maintain developmentally regulated MYC transcription, cell and tissue growth. Mammalian signaling networks are well known to co-ordinate transcriptional up- or down-regulation of many growth and cell cycle genes, particularly MYC. Ex vivo studies from the early 80s demonstrated that serum stimulation of mammalian tissue culture cells results in rapid activation of MYC transcription. Subsequently, extensive studies revealed that torsional stress and strain on dsDNA in the MYC promoter following initiation of MYC transcription results in melting of the double stranded FUSE (the Far Upstream Sequence Element/FUSE -1.7 kb upstream of the major MYC transcription start site) into ssDNA. Moreover, maximal activation of MYC transcription correlates with dissociation of RNA Pol II from the MYC TSS and recruitment of FBP1, which has been extensively characterized for specificity in binding the single stranded FUSE structure. The rapid release of RNA Pol II prior to the peak in MYC mRNA levels was associated with maximal enrichment for FBP1, consistent with FBP1 promoting RNA Pol II release to hyperactivate MYC transcription (Guo, 2016).

Based on these observations, and the current study in Drosophila, the following model is proposed for the action of Psi/FBP1 in activated MYC transcription. MED will first integrate the activity of MYC enhancers, stimulated by growth or developmental signals, with the general transcription factors (GTFs) and RNA Pol II to form the PIC. In a signaling environment conducive to MYC transcription, MED will recruit TFIIH and stabilise the PIC. MED will also bring TFIIH kinase activity into close proximity with the CTD of RNA Pol II, consistent with MED stimulating TFIIH-dependent CTD phosphorylation and RNA Pol II promoter clearance (Guo, 2016).

The increased RNA Pol II activity will result in conformational changes in the MYC promoter, including supercoiling to cause torsional stress and generation of the single-stranded FUSE, which binds FBP1/Psi. FBP1 can also directly interact with the XPB helicase subunit of TFIIH, and this interaction is required for formation of the promoter loop between RNA Pol II and FUSE. Psi is found in complex with the kinase module subunits of MED, thus it is predicted Psi/FBP1 will first interact with the preactivated MYC promoter, i.e., with the large MED complex still in residence. Structural changes in ssDNA following Psi/FBP1 loading will modulate promoter architecture further to facilitate exit of the CDK8 module, thus maximising MED-driven RNA Pol II activity and MYC transcription. At this stage it is not possible to make conclusions on whether Psi/FBP1 interacts directly with a given MED subunit, or indirectly via the TFIIH complex, however, the transition to a maximally activated MYC promoter is predicted to be be dependent on Psi/FBP1 (Guo, 2016).

Subsequently, FBP-Interacting Repressor (FIR/Hfp) will be recruited to the MYC promoter via binding to ssDNA, FBP1 and TFIIH to facilitate FBP1 exit, inactivation of RNA Pol II and return of MYC transcription to basal levels. Moreover, RNA interference studies suggest FIR is required for repression of MYC transcription (5) and is dysregulated in cancers with associated elevation of MYC. Previous Drosophila studies suggested the RRM protein with most similarity to FIR, Half Pint (Hfp), is essential for developmentally driven downregulation of dMYC transcription in vivo. Moreover, as observed for FIR, Hfp interacts with the XPB helicase component of the general transcription factor TFIIH to maintain a pool of engaged RNA Pol II on the MYC promoter, consistent with poised RNA Pol II being required to attenuate MYC transcription (Guo, 2016).

This analysis of the Drosophila protein interaction map revealed the XPB/Haywire subunit of TFIIH was not one of the 3488 bait proteins in the DPiM screen; nor was XPB/Haywire detected as one of the 4927 prey proteins with 0.8% (or less) false discovery. This was despite known interactors (including other TFIIH complex subunits such as CycH, Cdk7 and MAT1) being included as bait in the DPiM screen. Moreover, CycH, and Cdk7 only detected other CAK subunits (MAT1 and Cdk7 or CycH), but did not detect any of the core subunits (e.g. dXPB/Haywire). MAT1 as bait detected Cdk7, CycH and XPD from the core, but no other subunits. Thus, the DPiM is not saturating, in particular the core TFIIH subunits appear to be under represented, and further studies are required to establish whether Psi interacts with XBP/TFIIH (Guo, 2016).

These studies demonstrate nuanced mechanisms of MYC transcriptional regulation, requiring interaction between the MED complex and ssDNA binding proteins are essential for normal tissue growth. Further studies are required to determine whether human FBP1 also interacts with MED as it is predicted that this interaction will also modulate expression of the MYC oncogene. As even subtle increases in MYC expression (>2-fold) can promote the cell and tissue overgrowth fundamental to cancer initiation and progression, these observations will have implications for human disease (Guo, 2016).

The Drosophila splicing factor PSI is phosphorylated by casein kinase II and tousled-like kinase

Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors (Taliaferro, 2013).

Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein

Drosophila P-element somatic inhibitor protein (PSI) regulates splicing of the P-element transposase pre-mRNA by binding a pseudo-splice site upstream of the authentic splice site using four tandem KH-type RNA binding motifs. While the binding domains and specificity of PSI have been established, little is known about the contributions of each PSI KH domain to overall protein stability and RNA binding affinity. Using a construct containing only the RNA binding domain of PSI (PSI-KH03), a physiologically relevant point mutation was introduced into each KH domain of PSI individually and stability and RNA binding affinity of the resulting mutant proteins were measured. Although secondary structure, as measured by circular dichroism spectroscopy, is only subtly changed for each mutant protein relative to wild type, RNA binding affinity is reduced in each case. Mutations in the second or third KH domains of the protein are significantly more deleterious to substrate recognition than mutation of the outer (first and fourth) domains. These results show that despite the ability of a single KH domain to bind RNA in some systems, PSI requires multiple tandem KH domains for specific and high-affinity recognition of substrate RNA (Chmiel, 2006).

Structural basis of the interaction between P-element somatic inhibitor and U1-70k essential for the alternative splicing of P-element transposase

P-element transposition in Drosophila is regulated by tissue-specific alternative splicing of the P-element transposase pre-mRNA. In somatic cells, the P-element somatic inhibitor (PSI) protein binds to exon 3' UTR of the pre-mRNA and recruits U1 small nuclear ribonucleoprotein (snRNP) to the F1 pseudo-splice site. This abrogates binding of U1 snRNP to the genuine 5' splice site, thereby preventing excision of the third intron. Two homologous short sequences, referred to as the A and B boxes, near the C terminus of PSI bind to U1-70k protein within U1 snRNP. This study has mapped the AB box-binding site of U1-70k to a short proline-rich sequence at the C terminus. This NMR study shows that the B box forms an anti-parallel helical hairpin in which four highly conserved aromatic residues form a cluster on one face of the first helix. This hydrophobic cluster interacts extensively with the proline-rich region of the U1-70k protein (Ignjatovic, 2005).

Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila

To gain insight into splicing regulation, this study developed a microarray to assay all annotated alternative splicing events in Drosophila melanogaster, and the alternative splice events controlled by four splicing regulators: dASF/SF2, B52/SRp55, hrp48, and PSI were identified. The number of events controlled by each of these factors was found to be highly variable: dASF/SF2 strongly affects >300 splicing events, whereas PSI strongly affects only 43 events. Pairwise analysis also revealed many instances of splice site usage affected by multiple factors and provides the framework to understand the network controlling the alternatively spliced mRNA isoforms that compose the Drosophila transcriptome (Blanchette, 2005).

The KH-type RNA-binding protein PSI is required for Drosophila viability, male fertility, and cellular mRNA processing

Direct interactions between RNA-binding proteins and snRNP particles modulate eukaryotic pre-mRNA processing patterns to control gene expression. This study reports that the conserved U1 snRNP-interacting RNA-binding protein PSI is essential for Drosophila viability. A null PSI mutation is recessive lethal at the first-instar larval stage, and lethality is fully rescued by transgenes expressing the PSI protein. A mutant transgene that lacks the PSI-U1 snRNP-interaction domain restores viability but shows courtship behavior abnormalities and meiosis defects during spermatogenesis, resulting in a complete male sterility phenotype. Using cDNA microarrays, specific target mRNAs with altered expression profiles were identified in these mutant males. A subset of these transcripts is also found associated with PSI in endogenous immunopurified ribonucleoprotein complexes. One specific target, the hrp40/squid transcript, shows an altered pre-mRNA splicing pattern in PSI mutant testes. It is concluded that a functional association between the PSI protein and the spliceosomal U1 snRNP particle is required for normal Drosophila development and for the processing of specific PSI-interacting cellular transcripts. These results also validate the use of cDNA microarrays to characterize in vivo RNA-processing defects and alternative pre-mRNA splicing patterns (Labourier, 2002).

Modulation of P-element pre-mRNA splicing by a direct interaction between PSI and U1 snRNP 70K protein

P-element somatic inhibitor (PSI) is a KH domain-containing splicing factor highly expressed in Drosophila somatic tissues. This study has identified a direct association of PSI with the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle in somatic nuclear extracts. This interaction is mediated by highly conserved residues within the PSI C-terminal AB motif and the U1 snRNP-specific 70K protein. Through the AB motif, PSI modulates U1 snRNP binding on the P-element third intron (IVS3) 5' splice site and its upstream exonic regulatory element. Ectopic expression experiments in the Drosophila female germline demonstrate that the AB motif also contributes to IVS3 splicing inhibition in vivo. These data show that the processing of specific target transcripts, such as the P-element mRNA, is regulated by a functional PSI-U1 snRNP interaction in Drosophila (Labourier, 2001).

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro (Amarasinghe, 2001).

The alternative splicing factor PSI regulates P-element third intron splicing in vivo

Splicing of the Drosophila P-element third intron (IVS3) is inhibited in somatic cells, restricting transposase expression to the germ line. Somatic inhibition of IVS3 splicing involves the assembly of a multiprotein complex on a regulatory sequence in the IVS3 5' exon. The P-element somatic inhibitor protein (PSI) is a component of this ribonucleoprotein complex and is required for inhibition of IVS3 splicing in vitro. The soma-specific expression pattern of PSI suggests that its low abundance in the germ line allows IVS3 splicing. This study demonstrates that ectopic expression of PSI in the female germ line is sufficient to repress splicing of an IVS3 reporter transgene. It was also shown that IVS3 splicing is activated in somatic embryonic cells in the presence of an antisense PSI ribozyme. These results support the model that PSI is a tissue-specific regulator of IVS3 splicing in vivo (Adams, 1997).

Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing

PSI is an RNA-binding protein involved in repressing splicing of the P element third intron in Drosophila somatic cell extracts. PSI produced in bacteria restores splicing inhibition to an extract relieved of inhibitory activity, indicating that PSI plays a direct role in somatic inhibition. Sequence analysis of cDNAs encoding PSI reveals three KH RNA-binding domains, a conserved motif also found in the yeast splicing regulator MER1. Notably, PSI is expressed highly in somatic embryonic nuclei but is undetectable in germ-line cells. In contrast, hrp48, another protein implicated in somatic inhibition, is found in the nucleus and cytoplasm of both tissues. The splicing inhibitory properties and soma-specific expression of PSI may be sufficient to explain the germ-line-specific transposition of P elements (Siebel, 1995).

Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. This study has purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). The 50-kD protein was identified as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and hrp48 was shown to recognize specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48 (Siebel, 1994).

Search PubMed for articles about Drosophila Psi

Adams, M. D., Tarng, R. S. and Rio, D. C. (1997). The alternative splicing factor PSI regulates P-element third intron splicing in vivo. Genes Dev 11(1): 129-138. PubMed ID: 9000056

Amarasinghe, A. K., MacDiarmid, R., Adams, M. D. and Rio, D. C. (2001). An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element. RNA 7(9): 1239-1253. PubMed ID: 11565747

Blanchette, M., Green, R. E., Brenner, S. E. and Rio, D. C. (2005). Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 19(11): 1306-1314. PubMed ID: 15937219

Chmiel, N. H., Rio, D. C. and Doudna, J. A. (2006). Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein. RNA 12(2): 283-291. PubMed ID: 16428607

Foley, L., Ling, J., Joshi, R., Evantal, N., Kadener, S. and Emery, P. (2019). Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing. Elife 8. PubMed ID: 31702555

Goodliffe, J. M., Wieschaus, E. and Cole, M. D. (2005). Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila. Genes Dev 19(24): 2941-2946. PubMed ID: 16357214

Guo, L., Zaysteva, O., Nie, Z., Mitchell, N. C., Amanda Lee, J. E., Ware, T., Parsons, L., Luwor, R., Poortinga, G., Hannan, R. D., Levens, D. L. and Quinn, L. M. (2016). Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth. Nucleic Acids Res 44(16): 7646-7658. PubMed ID: 27207882

Ignjatovic, T., Yang, J. C., Butler, J., Neuhaus, D. and Nagai, K. (2005). Structural basis of the interaction between P-element somatic inhibitor and U1-70k essential for the alternative splicing of P-element transposase. J Mol Biol 351(1): 52-65. PubMed ID: 15990112

Labourier, E., Adams, M. D. and Rio, D. C. (2001). Modulation of P-element pre-mRNA splicing by a direct interaction between PSI and U1 snRNP 70K protein. Mol Cell 8(2): 363-373. PubMed ID: 11545738

Labourier, E., Blanchette, M., Feiger, J. W., Adams, M. D. and Rio, D. C. (2002). The KH-type RNA-binding protein PSI is required for Drosophila viability, male fertility, and cellular mRNA processing. Genes Dev 16(1): 72-84. PubMed ID: 11782446

Lim, C. and Allada, R. (2013). ATAXIN-2 activates PERIOD translation to sustain circadian rhythms in Drosophila. Science 340(6134): 875-879. PubMed ID: 23687047

Martin Anduaga, A., Evantal, N., Patop, I. L., Bartok, O., Weiss, R. and Kadener, S. (2019). Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila. Elife 8. PubMed ID: 31702556

Schuijers, J., Manteiga, J. C., Weintraub, A. S., Day, D. S., Zamudio, A. V., Hnisz, D., Lee, T. I. and Young, R. A. (2018). Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism. Cell Rep 23(2): 349-360. PubMed ID: 29641996

Shakhmantsir, I., Nayak, S., Grant, G. R. and Sehgal, A. (2018). Spliceosome factors target timeless (tim) mRNA to control clock protein accumulation and circadian behavior in Drosophila. Elife 7. PubMed ID: 30516472

Siebel, C. W., Kanaar, R. and Rio, D. C. (1994). Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes Dev 8(14): 1713-1725. PubMed ID: 7958851

Siebel, C. W., Admon, A. and Rio, D. C. (1995). Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing. Genes Dev 9(3): 269-283. PubMed ID: 7867926

Taliaferro, J. M., Marwha, D., Aspden, J. L., Mavrici, D., Cheng, N. E., Kohlstaedt, L. A. and Rio, D. C. (2013). The Drosophila splicing factor PSI is phosphorylated by casein kinase II and tousled-like kinase. PLoS One 8(2): e56401. PubMed ID: 23437125

Wang, Q., Taliaferro, J.M., Klibaite, U., Hilgers, V., Shaevitz, J.W. and Rio, D.C. (2016). The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27114556

Zaytseva, O., Mitchell, N. C., Guo, L., Marshall, O. J., Parsons, L. M., Hannan, R. D., Levens, D. L. and Quinn, L. M. (2020). Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila. Development 147(11). PubMed ID: 32527935

Zhang, Z., Cao, W. and Edery, I. (2018). The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila. Sci Rep 8(1): 1872. PubMed ID: 29382842

date revised: 30 October 2020

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