Alternative splicing is extensive in both species, supporting the argument that both species benefit from expanded proteomes generated from alternative splicing. However, genome analysis suggests that there are significant differences in the weight of the mechanisms by which alternative splicing can be induced. In Drosophila, intron length is a major determinant in promoting alternative splicing patterns. In the human, additional mechanisms of controlling alternative splicing may have gained more influence on intron expansion to maintain balanced levels of alternative splicing (Fox-Walsh, 2005).

G3BP RNA-binding proteins are important components of stress granules (SGs). This study analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, it was found that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. RIN was shown to be associated with polysomes and evidence was provided for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. It is proposed that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules (Laver, 2020).

Post-transcriptional regulation (PTR) plays a key role in the control of gene expression in all cell types. PTR is achieved by RNA-binding proteins (RBPs) and small RNAs, such as microRNAs (miRNAs), which act as specificity factors that modulate the interaction of mRNAs with the cellular machinery that localizes, translates, and degrades mRNAs (Laver, 2020).

PTR is particularly important in early animal embryos, where maternally provided mRNAs and proteins control developmental events prior to transcriptional activation of the embryo's genome. In several model animals, including Drosophila, the transfer of control from maternal products to those synthesized by the embryo's own genome -- the maternal-to-zygotic transition (MZT) -- is very rapid, occurring over a matter of hours, and thus facilitating studies of the mechanisms and functions of PTR. For example, it has been shown that the Drosophila RBP Smaug (SMG), which binds to specific stem-loop structures in its target mRNAs to repress their translation and trigger their degradation, is essential for repression and clearance of hundreds of maternal mRNAs and for timely activation of the zygotic genome. SMG is not the only negative regulator of maternal transcripts in Drosophila; additional RBPs (e.g., brain tumor) or miRNAs (e.g., miR-309) function in maternal mRNA clearance (Laver, 2020).

PTR also serves as a rapid response to cellular stress. Under stress conditions, cells shut down translation of many mRNAs while upregulating transcription and/or translation of sets of protein chaperones that maintain basal cellular integrity. Repression occurs, at least in part, in membraneless organelles known as stress granules (SGs). SGs are thought to contain transcripts that are stalled in translation initiation, and recent global analyses have shown that SGs are enriched for long transcripts (Laver, 2020).

An important component of SGs is the RBP G3BP, which is conserved throughout eukaryotes. Mammals have two genes, G3BP1 and G3BP2, whereas in Drosophila there is a single gene, Rasputin (rin). In human cells, G3BPs are necessary for SG formation, and if overexpressed, they are sufficient to induce SGs even in the absence of stress. RIN is necessary for SG formation in the Drosophila S2 tissue culture cell line, and although RIN or G3BP overexpression can induce SGs in human cells, this is not the case in S2 cells. RIN and G3BPs interact with several of the same protein partners under both stress and non-stress conditions; these include Caprin (CAPR), FMRP (FMR1 in Drosophila), and UPA2 (Lingerer [LIG] in Drosophila) (Laver, 2020 and references therein).

The roles of RIN/G3BPs in unstressed cells have received less attention than their roles upon stress. Multiple functions have been attributed to G3BPs, including transcript destabilization and repression (e.g., c-myc, BART, CTNNB1, PMP22, HIV-1, and miR-1), transcript stabilization (e.g., Tau and SART3), subcellular transcript localization (e.g., Twist1), and transcript sequestration into virus- induced foci (e.g., HIV-1). In Drosophila, mutations in the rin gene cause severe defects in oogenesis, mutant females lay few eggs, and those that are laid fail to hatch. rin mutations can also result in tissue patterning and growth defects. Despite RIN's essential function in the fly life cycle, there have been no analyses of the RIN-bound transcriptome or RIN's global role in gene regulation, nor are the molecular mechanisms that underlie RIN function known (Laver, 2020).

To better understand the function of RIN/G3BP in unstressed cells, a global analyses of the RIN-associated proteome and transcriptome was carried out in early Drosophila embryos. Using an anti-RIN synthetic antibody that was isolated from a phagedisplayed library of fragments antigen binding (Fab), RIN was immunoprecipitated and then mass spectrometry (IP-MS) was carried out to identify RIN's partner proteins. Interactions were found with several RBPs previously shown to interact with G3BP/RIN (e.g., CAPR, FMR1, and LIG), consistent with IP of a biologically relevant RIN-containing complex. RNA-dependent interactions with RIN were found for both small and large ribosomal subunit proteins, suggesting that RIN may be polysome associated, a fact that that was confirmed using polysome gradients. By coimmunoprecipitating RIN together with bound mRNAs followed by microarray analysis, hundreds of in vivo target transcripts were identified in embryos that are characterized by two features: they are short and enriched for a binding motif that was previously identified in vitro. RIN-associated mRNAs are enriched for Gene Ontology (GO) terms for core components of the transcription, splicing, and translation machinery as well as of mitochondria. RIN's endogenous targets in early embryos are more stable and more highly translated than co-expressed unbound transcripts. Their shorter length and higher rates of translation contrast with the behavior of mRNAs associated with SGs. Consistent with a role for RIN as a positive regulator of transcript stability, in rin mutants, the abundance of several highly bound target mRNAs is reduced relative to controls. Using a heterologous RNA-binding domain to tether RIN, G3BP1, or G3BP2 to a luciferase reporter mRNA in S2 tissue culture cells, it was confirmed that RIN/G3BP increases the stability and/or translation of bound transcripts in the absence of stress (Laver, 2020).

The data support a conserved function for G3BP proteins as potentiators of the translation and stability of their target transcripts. It is speculated that stress-dependent recruitment of G3BPs/RIN into SGs may serve as a mechanism to downregulate gene expression both directly, by removing RIN from its endogenous target mRNAs, as well as indirectly, through reduced transcription, splicing, and translation (Laver, 2020).

Two features, short transcript length and the motif bound by the RIN RRM in vitro, are predictive of RIN binding. That short transcripts in general might be more likely to contain the motif is excluded by the fact that RIN's target mRNAs are enriched for the motif compared with length-matched, co-expressed, unbound mRNAs. Thus, it is proposed that each feature separately contributes to RIN target mRNA binding, with shortness playing a greater role than the motif (Laver, 2020).

Although it is unclear how RIN is able to measure transcript length, there is a precedent for the differential behavior and regulation of short versus long mRNAs. For example, in general, short mRNAs are more highly translated than long mRNAs, and this is thought to reflect the fact that short mRNAs have a higher affinity for the cap-binding complex. It has been proposed that this higher affinity is related to the possibility that short mRNAs are able to form a closed-loop structure more readily than longer mRNAs. It is noted that a recent study that calls the closed-loop model into question was unable to test short mRNAs because of technical limitations. Thus, even if long mRNAs do not form a stable closed loop, it remains possible that short mRNAs do (Laver, 2020).

The ribosome-associated protein RACK1 has been shown to be required for the efficient translation of short but not long mRNAs. Although this study has shown that RIN is associated with polysomes in early embryos, Drosophila RACK1 is not on the list of RIN protein interactors and neither is there a significant overlap between the RIN protein interaction network and a set of protein interactors previously identified for RACK1. Thus, the mechanisms by which RIN recognizes short mRNAs may differ from those that have been identified previously (Laver, 2020).

Another striking feature of RIN-bound mRNAs is that they are depleted for SREs, the binding sites of SMG, which destabilizes and translationally represses its target mRNAs. This could suggest that the high translational efficiencies and stability of RIN-target mRNAs is simply a result of a lack of SREs. However, this study has provided evidence that RIN can exert its effects in situations where the SMG protein is not present (i.e., during oogenesis, in embryos older than 3 h, and in S2 cells). Thus, it is concluded that most RIN-bound mRNAs in the early embryo are upregulated directly by RIN and indirectly through a lack of SREs (Laver, 2020).

Because the target mRNAs of RIN are enriched for GO terms related to multiple levels of the core gene expression machinery-transcription, splicing, and translation-RIN may directly potentiate the expression of its bound target transcripts and, in so doing, also indirectly upregulate gene expression globally. As an example of how RIN/G3BP's direct and indirect effects might converge on the same cellular process, the production of cytoplasmic ribosomes is considered. Metabolic labeling with radioactive amino acids has shown that cytoplasmic ribosomal protein (cRP) synthesis increases after fertilization, peaks at 3-4 h, and subsequently decreases. Recent ribosome occupancy-based measurements have confirmed that the translational efficiency of cRP mRNAs increases in early embryos relative to mature oocytes. This study has shown that RIN potentiates target mRNA stability and translation and that cRP mRNAs are highly enriched among RIN's targets; thus, the potentiation of cRP mRNAs is expected to be a direct effect of RIN (Laver, 2020).

cRP mRNAs are also regulated by their conserved 50-terminal oligopyrimidine (50TOP) motifs. Two RBPs, La and Larp1, have been implicated in regulation of the stability and/or translation of 50TOP mRNAs by this motif. This study shows that the Drosophila orthologs of both of these RBPs-LA and LARP-associate with RIN in an RNA-dependent manner. This could reflect the fact that LA, LARP, and RIN co-bind cRP mRNAs and, thus, that this class of mRNAs is subject to multiple direct mechanisms that potentiate its expression. Noteworthy is the fact that, of the RIN target mRNAs, only the cRP transcripts carry 50TOP motifs and, as such, they represent a distinct class of mRNAs (Laver, 2020).

Human Pumilio (hPUM see Drosophila Pumilio) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear. This study reports how the repeats contribute to overall RNA binding. The first step was to prepare three mutants in which R1' and/or R8' were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1' contributed more than R8', unlike Drosophila PUM. It was further investigated which amino acid residues of R1' or R8' were responsible for RNA binding. With detailed analysis of the protein tertiary structure, this study found a hydrophobic core in each of the repeats. All hydrophobic amino residues in each core to alanine were mutated during this process. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1' had a significant influence. In the present study, it was demonstrated that the flanking R1' and R8' repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1'-R1 interactions may stabilize the whole hPUM structure (Nakamura, 2021).

The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). It has been shown previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. This study determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. interactions of Glo qRRMs were probed with TCEI_III RNA using NMR paramagnetic relaxation experiments. The in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated (Warden, 2023).

Specific recognition of target RNAs by RNA-binding proteins is essential for gene control and often achieved by modular combination of multiple RNA-binding domains. Tandem RNA-binding domains may arise from duplication to allow recognition of longer linear RNA sequences or more complex structures than a single domain can accomplish. For example, the tandem zinc fingers of Tristetraprolin each recognize a UAUU sequence within the UUAUUUAUU AU-rich element (ARE). Duplicated domains also may diverge to recognize distinct sequence or structural elements. In the case of HuD protein, its two N-terminal RNA recognition motif (RRM) domains both recognize UU elements in AREs, but RRM1 also interacts with four additional 5' flanking nucleotides. Although amino acid sequences of RNA-binding domains can indicate whether they retain the characteristic RNA recognition features of that domain family, it remains impossible to predict most domain-specific variations. Hence, the specificity of RNA-binding domains must be determined experimentally. Each study expands knowledge of sequence features that drive specificity and improves prediction methods to identify target RNAs (Warden, 2023).

During Drosophila development, translational control of nanos (nos) mRNA is essential for the formation of the anterior-posterior body axis of the embryo. This process begins in the ovary when nos mRNA is transferred to oocytes from nurse cells during late oogenesis and translationally repressed until it is localized to the posterior. The translational repression of unlocalized nos mRNA is mediated by a translational control element (TCE) in the 3' UTR of nos mRNA. nos TCE RNA is composed of one stem (TCEI) and two stem loop structures (TCEII and TCEIII). During oogenesis, binding of TCEI and TCEIII by the protein Glorund (Glo) controls Nos expression. This protein:RNA interaction is essential to repress unlocalized nos mRNA during oogenesis, which ultimately ensures the formation of functional anterior structures in the fly embryo. Repression of nos initiated by Glo during oogenesis is also maintained in the early embryo by another RNA-binding protein, Smaug, which recognizes TCE stem loop II (Warden, 2023).

Glo is an RNA-binding protein belonging to the heterogenous nuclear ribonucleoprotein F/H (hnRNP F/H) family. Mammalian hnRNP F and H are predominantly nuclear and best known as regulators of alternative splicing. Additionally, hnRNP F and H1 are abundant in regenerating axons, where they bind to mRNAs involved in axonal growth and are thought to regulate their axonal transport and/or translation. Notably, mammalian hnRNP F and H proteins are upregulated in a variety of cancers and are thought to contribute to pathogenicity in part by altering translation. Like hnRNPs F/H, Glo contains three tandem quasi-RNA recognition motifs (qRRMs). It was previously demonstrated that the Glo qRRMs are multifunctional, each capable of binding to G-tract and structured UA-rich features of the nos TCE. The G-tract sequence is found in TCEI and the structured UA-rich motif in TCEIII. Both features are essential for TCE function and Glo recognition, yet the affinity for G-tract binding is substantially higher than UA-rich motif binding. Disruption of either RNA motif results in modestly increased Nos protein, but mutation of both RNA motifs dramatically increases Nos protein levels. Moreover, engineered mutations that preferentially disrupt Glo qRRM binding to either the G-tract or UA-rich motif demonstrate that nos repression requires both modes of Glo-TCE recognition in vivo. How the tandem Glo qRRMs combine to recognize the G-tract and UA-rich motifs of TCE RNA remained unanswered (Warden, 2023).

This study presents a high-resolution solution structure of an RNA containing the elements recognized by Glo: nos TCEI and TCEIII, which is called in this study TCEI_III. The divide-and-conquer method, a well-established and effective approach combining structural biology methods, was used to build a larger macromolecular model from high-resolution structures of its components. This divide-and-conquer method allowed this study to avoid the technical difficulties of crystallizing structured RNAs without engineered crystal contacts such as tetraloop/tetraloop insertions. First a solution structure of TCEIII RNA was determined by NMR. Then the TCEIII structure along with NMR data for TCEI_III RNA and overall shape information from small-angle X-ray scattering (SAXS) were used to produce the hybrid TCEI_III model. This model suggested that more than one Glo qRRM is necessary for TCE recognition, and in vivo experiments indicated that a minimum of two qRRMs is sufficient for nos repression. Furthermore, NMR paramagnetic relaxation experiments (PREs) were used to probe the interactions between the G-tract and UA-rich motifs of TCE RNA and individually-labeled Glo qRRMs. Our in vitro and in vivo data are consistent with a model where the Glo qRRMs are indeed multifunctional and interchangeable for recognition of nos TCE G-tract or UA-rich motifs (Warden, 2023).

Combinations of RNA recognition modules generate the diverse specificities of RNA-binding proteins needed to control gene expression. RRM or KH domains often occur in tandem and together generate distinct RNA recognition specificity to select target mRNAs. Although much information is available about the specificities of RNA-binding domains, including high-throughput screening of sequence specificity, there relatively little information on how multiple RNA-binding modules bring their individual specificities to RNA target selection. This study demonstrates that at least two of the three multifunctional Glo qRRMs are required to recognize the nos TCE RNA, one for the TCEIII UA-rich stem and one for the TCEI G-tract. Therefore, the efficiency of having two RNA-binding modes in one qRRM is not utilized: one domain does not simultaneously recognize both RNA features. It was also found that the three qRRMs are interchangeable: any two of the three Glo qRRMs are sufficient in vivo to repress nos translation and maintain viability. Although qRRMs 1 and 2 are closely spaced with 8 residues between the C-terminal α helix of qRRM1 and the N-terminal β strand of qRRM2, this length linker appears sufficient to bridge the distance between G-tract and UA-rich elements. Alternatively, the C-terminal α helix of qRRM1 could unfold upon RNA binding, as seen for the RRM of Nop15 when it binds to pre-rRNA. Finally, it does not matter which RNA feature is recognized by the two qRRMs. This striking redundancy of the Glo qRRMs seems excessive, as some specialization would have been expected to have evolved (Warden, 2023).

Several evolutionary benefits are suggested from having three redundant multifunctional qRRMs in a single protein. (i) Functional redundancy protects crucial gene regulation. Repression of nos translation during oogenesis is essential to ensure correct anterior patterning in the embryo, yet mutation of one qRRM avoids a biological disruption as nos expression is normal in embryos. Similarly, although the target RNA(s) are not known, Glo with two intact qRRMs supports viability to adulthood. (ii) Redundancy gives Glo the potential to recognize a variety of target RNAs with different combinations of G-tract and UA-rich features. In addition, the G-tracts may be either single-stranded or base-paired. Previous work has demonstrated that Glo qRRMs bind to short single-stranded G-tracts, and this study observed visible imino proton resonances for the G-tract sequence (G87, G88, and G89) in the TCEI_III RNA, indicating that the G-tract remains base paired in the presence of Glo. Therefore, Glo qRRMs appear to differ from hnRNP F qRRMs that disrupt RNA structures when binding to G-tracts. Although the in vivo and in vitro evidence indicates the importance of base pairing for TCE activity, it will require additional structural studies to determine how Glo qRRMs specifically recognize duplex RNAs. (iii) Functionally redundant qRRMs with different spacing between the qRRMs adds additional opportunities for distinct RNA recognition. The linker between Glo qRRMs 1 and 2 is short (∼8 aa) whereas qRRMs 2 and 3 are separated by a long linker (∼240 aa). Although the distance between qRRMs does not affect nos regulation as any two qRRMs are sufficient, the spacing may alter specificity for other RNAs. For example, qRRMs 1 and 2 would require RNAs with G-tract and/or UA-rich motifs near each other whereas the long linker between qRRMs 2 and 3 should make that two-domain combination less restrictive in arrangement of G-tract and/or UA-rich motifs in target RNAs. To appreciate the range of specificity and roles of its multifunctional qRRMs, additional RNAs that are controlled by Glo will need to be identified (Warden, 2023).

Adding to its multifunctionality, Glo also binds to other proteins. Glo was recently shown to interact with Drosophila Fragile X mental retardation protein (dFMRP) to stall ribosomes and decrease the translation elongation rate on nos mRNA. Although this study has not identified differences in RNA recognition, Glo qRRMs do have different protein binding specificity. Glo interacts with dFMRP via qRRM2, but qRRM1 and qRRM3 do not directly interact with dFMRP. It is tempting to speculate that interaction between qRRM2 and dFMRP alters RNA recognition, and that other protein interactions could modify RNA recognition by the other qRRMs to generate specialization (Warden, 2023).