InteractiveFly: GeneBrief

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The NineTeen Complex (NTC), also known as Pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. This study shows that NTC-subunit Salsa, the Drosophila orthologue of human RNA helicase Aquarius (CG31368), is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken. Germ line depletion of Salsa and splice site mutations within gurken first intron both impair adult female fertility and oocyte dorsal-ventral patterning due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether these results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR (Rathore, 2020).

The spliceosome is a highly dynamic molecular machine, composed of five small nuclear ribonucleoproteins (snRNPs) that sequentially associate to the precursor mRNA (pre-mRNA) during the splicing reaction. Each snRNP (U1, U2, U4, U5, and U6) contains a U-rich snRNA and a unique group of proteins. Although spliceosome assembly is ordered (U1 > U2 > U4/U5/U6 > NineTeen Complex), the splicing reaction is without an apprarent irreversible and/or rate-limiting step, with commitment to splicing progressively increased as snRNPs and NTC bind to the pre-mRNA (Rathore, 2020).

The spliceosomal NineTeen Complex (NTC), also known as Pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for efficient pre-mRNA splicing (Hogg, 2010; Chanarat, 2013). NTC composition is dynamic and comprises a subset of conserved core subunits and many transiently associated ones (Hogg, 2010). NTC associates with the spliceosome during its activation and just before the first transesterification (Hogg, 2010). Interestingly, NTC also has a significant role in the crosstalk between transcription, cotranscriptional processing of the nascent RNA, and DNA repair, as distinct NTC subunits have been reported to be important for transcriptional elongation and genomic stability. Human NTC-subunits PRP19, XAB2, and CDC5L are important for transcriptional elongation, transcription-coupled DNA repair, and activation of the ATM-related (ATR)-dependent DNA damage checkpoint. RNA Polymerase II (RNA Pol II) also promotes cotranscriptional splicing activation through the recruitment of NTC (David et al. 2011) (Rathore, 2020).

Human Aquarius (AQR) (also known as intron-binding protein 160, IBP160) is an ATP-dependent RNA helicase that associates with NTC during spliceosome activation and formation of the activated B complex (BACT). AQR binds to introns independently of sequence, but usually upstream of the branch-site (BS) and close to the associated U2 snRNP SF3a and SF3b proteins, being essential for intron-binding complex formation and efficient splicing. AQR has also been suggested to be important for deposition of the exon junction complex (EJC) during the splicing reaction and formation of intron-encoded snoRNAs, suggesting it regulates the cross-talk between splicing and other RNA processing events (Rathore, 2020).

Splicing during Drosophila early embryonic development is notably sensitive to mutations in NTC-subunit Fandango (Guilgur, 2014), suggesting differential requirements of NTC during development (Martinho, 2015). To test this possibility, it was decided to investigate the role of other NTC-subunits during Drosophila oogenesis and early embryonic development. Focused of initial work was placed on uncharacterized gene CG31368, which encodes the Drosophila ortholog of human Aquarius. Since there is already a nonrelated Drosophila protease named aquarius (CG14061), CG31368 was renamed salsa. The working hypothesis is that salsa, similar to its Caenorhabditis elegans ortholog emb-4 (Akay, 2017), is likely to have important developmental functions (Rathore, 2020).

During Drosophila oogenesis, gurken mRNA localizes to the posterior cortex of the developing oocyte and Gurken signal is restricted to the underlying posterior follicle cells. In response to a signal from the posterior follicle cells, there is a considerable reorganization of the cytoskeleton and a microtubule-dependent migration of the oocyte nucleus to the anterior cortex. The anteriorly localized nucleus defines the dorsal-anterior region and provides the first detectable dorsal-ventral (D/V) asymmetry of the oocyte, with the expression of both gurken mRNA and protein restricted to the cytoplasmic perinuclear region of the oocyte (Rathore, 2020).

gurken mRNA is transcribed in the supporting nurse cells and actively transported to the dorsal-anterior region of the oocyte by a dynein-mediated transport. The oocyte dorsal-anterior localization of gurken mRNA relies on multiple elements localized to the transcript 5' UTR, 3' UTR and open-reading frame. Although this localization is crucial for its efficient translation, the precise contribution of each element for RNA localization is still a matter of debate (Rathore, 2020).

D/V patterning of the developing Drosophila egg is dependent on the dorsal-anterior localization of Gurken during mid-oogenesis. Gurken is the ligand for the Epidermal growth factor receptor (Egfr) that locates to the apical surface of follicle cells that surround the developing oocyte. Activation of Egfr modifies the cell fate of the dorsal follicle cells and restricts the formation of Spätzle ligand to the ventral region of the oocyte, which is essential for normal morphogenesis of the eggshell dorsal appendages (Rathore, 2020).

This study found that Salsa, the Drosophila ortholog of AQR, is rate-limiting for efficient splicing of a subset of small first introns, including the first intron of gurken. Consistent with the functional relevance of gurken splicing defects, mutations within the splice sites of the first intron of gurken impair the function of this gene. Female germline depletion of Salsa and splice mutations within gurken first intron were both associated to a decrease in female fertility, significant D/V patterning defects of the eggshell and abnormal expression of Gurken during oogenesis. Supporting causality, expression of a gurken construct without its first intron suppressed the female fertility and D/V patterning defects observed after Salsa depletion. Altogether these results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA (Rathore, 2020).

Oocyte dorsal-anterior localization of gurken mRNA relies on multiple elements localized to the transcript 5'UTR, 3'UTR and open-reading frame, yet the relative importance of each element for mRNA localization is still unclear. The 5' and 3'-UTRs of gurken were reported to be required for dorsal-anterior localization of gurken transcript. Furthermore, and using a genomic gurken construct with a lacZ reporter inserted within the gene open-reading frame, it was shown that whereas gurken 5'UTR is required for transcript oocyte accumulation, its coding region and 3'UTR are necessary for its posterior and dorsal-anterior localization. Nevertheless, it was recently reported, using an oocyte injection assay, that a small stem-loop located within the open-reading frame was necessary and sufficient for gurken transcript localization (Rathore, 2020).

The results show that efficient splicing of the first intron of gurken is required for mRNA dorsal-anterior localization and dorsal-ventral patterning. This is most likely because retention of the first intron impairs the secondary RNA structure of gurken 5'UTR, and the function of a closely located RNA element important for its localization. Splicing of the first intron of gurken is also likely to facilitate Gurken protein expression, as deletion of the first intron of gurken suppresses the dorsalization phenotype associated with increased copy number of gurken gene without affecting the levels of gurken mRNA. The results therefore fully support the role of gurken 5'UTR in mRNA localization within the oocyte, and strongly suggest that Salsa-dependent splicing of the first intron of gurken mRNA is important for the correct expression and function of this gene (Rathore, 2020).

The precise function of human Aquarius in splicing is still poorly understood. This RNA helicase is recruited to the spliceosome as a pentameric complex known as intronbinding complex (IBC), which also contains hSyf1 (also known as Xab2), hIsy1, CypE, and CCDC16 (De, 2015). Coimmunoprecipitation experiments suggest a large interaction interface between IBC and U2 snRNP, within the activated spliceosome (Bact stage) and just before the first splicing reaction. Although Aquarius ability to bind and hydrolyze ATP is important for spliceosome activation and splicing efficiency, the role of its RNA unwinding activity is less clear (Rathore, 2020).

This work has identified a small subset of introns whose splicing is particularly sensitive to depletion of Salsa (the Drosophila ortholog of human Aquarius). The fact that splicing was only affected in a small number of introns is consistent with the observation that immunodepletion of human Aquarius from nuclear extracts only weakly impaired splicing in vitro (De, 2015). This suggests that although this RNA helicase is apparently not critical for overall splicing, during female gametogenesis there is a subset of introns whose efficient removal relies on the function of this enzyme (Rathore, 2020).

Analysis of the introns whose splicing was sensitive to Salsa depletion showed a clear bias for small first introns with weak 3'splice sites, independently of their distance to the transcription start site (TSS), 5'splice site strength and GC content. The bias for small introns suggests that Salsa is mostly rate-limiting when introns are recognized by intron definition (Pai, 2017), where the initial pairing between U1 and U2 snRNPs occurs across the intron. Furthermore, the bias for introns with weak 3'splice sites is in accordance with the extensive interaction between IBC and U2 snRNP in the activated spliceosome, and implies that depletion of Salsa is likely to impair, at least in a subset of introns, U2 snRNP function during splicing. The absence of any detectable bias for short distances between the TSS and 5'splice site, when evaluating affected and control first introns, or any bias for weak 5'splice site strength, suggests that Salsa is not likely rate-limiting for Cap-Binding Complex-mediated splicing (Rathore, 2020 and references therein).

Drosophila first introns are more likely to be cotranscriptionally retained than internal and terminal introns. This is not consistent with the kinetic competition model, where the fastest processes are the ones most likely to occur, suggesting additional constraints to first intron splicing. Although the precise nature of such constraints is still poorly understood, binding of transcriptional initiation factors to the 5'splice site-associated U1snRNP potentially restricts splicing efficiency, as it might impair the initial pairing between U1 and U2 snRNPs. The current working hypothesis is that Salsa is required for splicing of small first introns with weak 3'splice site because this enzyme facilitates U2 snRNP function, minimizing the interference effect of transcriptional initiation factors on splicing. Future work will help define the function of this RNA helicase and its contribution for differential gene expression during development (Rathore, 2020).

Search PubMed for articles about Drosophila Salsa

Akay, A., Di Domenico, T., Suen, K. M., Nabih, A., Parada, G. E., Larance, M., Medhi, R., Berkyurek, A. C., Zhang, X., Wedeles, C. J., Rudolph, K. L. M., Engelhardt, J., Hemberg, M., Ma, P., Lamond, A. I., Claycomb, J. M. and Miska, E. A. (2017). The helicase Aquarius/EMB-4 is required to overcome intronic barriers to allow nuclear RNAi pathways to heritably silence transcription. Dev Cell 42(3): 241-255 e246. PubMed ID: 28787591

Chanarat, S. and Strasser, K. (2013). Splicing and beyond: the many faces of the Prp19 complex. Biochim Biophys Acta 1833(10): 2126-2134. PubMed ID: 23742842

De, I., Bessonov, S., Hofele, R., dos Santos, K., Will, C. L., Urlaub, H., Luhrmann, R. and Pena, V. (2015). The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes. Nat Struct Mol Biol 22(2): 138-144. PubMed ID: 25599396

Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181. PubMed ID: 24755291

Haselbach, D., Komarov, I., Agafonov, D. E., Hartmuth, K., Graf, B., Dybkov, O., Urlaub, H., Kastner, B., Luhrmann, R. and Stark, H. (2018). Structure and conformational dynamics of the human spliceosomal B^act Complex. Cell 172(3): 454-464 e411. PubMed ID: 29361316

Hogg, R., McGrail, J. C. and O'Keefe, R. T. (2010). The function of the NineTeen Complex (NTC) in regulating spliceosome conformations and fidelity during pre-mRNA splicing. Biochem Soc Trans 38(4): 1110-1115. PubMed ID: 20659013

Martinho, R. G., Guilgur, L. G. and Prudencio, P. (2015). How gene expression in fast-proliferating cells keeps pace. Bioessays 37(5): 514-524. PubMed ID: 25823409

Pai, A. A., Henriques, T., McCue, K., Burkholder, A., Adelman, K. and Burge, C. B. (2017). The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture. Elife 6. PubMed ID: 29280736

Rathore, O. S., Silva, R. D., Ascensao-Ferreira, M., Matos, R., Carvalho, C., Marques, B., Tiago, M. N., Prudencio, P., Andrade, R. P., Roignant, J. Y., Barbosa-Morais, N. L. and Martinho, R. G. (2020). NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning. Rna. PubMed ID: 32963109

date revised: 10 February 2021

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