Drosophila MFAP1 Is Required for Pre-mRNA Processing and G2/M Progression

Abstract

The mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions (Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component (Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938 -2946), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G 2 /M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/ cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G 2 /M arrest might be a general consequence of interfering with spliceosome function.Splicing of pre-mRNAs is a catalytic reaction that involves two successive trans-esterification steps. This process is carried out by a highly conserved ribonucleoprotein complex, called the spliceosome. The spliceosome consists of five snRNAs (U1, U2, U4, U5, and U6) and more than 200 proteins, making it one of the largest and most complex molecular machines studied (1). Spliceosome assembly is a sequential process that is initiated by the recruitment of the U1 small nuclear ribonucleoprotein (snRNP) 2 to the 5Ј-splice donor site to form a "commitment complex" ("E complex" in mammals) (3, 4). Subsequently, recruitment of the U2 snRNP to the branch site by the U2 auxiliary factor of 65 kDa (U2AF65) generates the "pre-spliceosome" ("A complex" in mammals) (5-8). A preformed U4/U6.U5 tri-snRNP unit then joins the U1-U2-pre-mRNA complex to form the "complete spliceosome" ("B complex" in mammals). To activate the complete spliceosome several conformational rearrangements must take place (reviewed in Ref.9). These include unwinding of base pairings between U1 and the 5Ј-splice site and between U6 and U4, as well as the formation of a new base pair interaction between U5 and U6 snRNPs and the 5Ј-splice site and U6 and U2. As a result, U1 and U4 snRNPs are released and an active spliceosome is formed (10, 11). The yeast RNA helicases Prp28p and Brr2p are both implicated in the structural rearrangements that take place during the activation step of the spliceosome (11). Prp28p is thought to be important for the unwinding of base pairings between U1 and the 5Ј-splice site (12) and Brr2p is implicated in unwinding the U4/U6...

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

“…2B, lower panel). However, similar to previous reports, the stg pre-mRNA level was not detectable by quantitative RT-PCR; this was suggested to be attributable to low levels or instability of the pre-mRNA (Andersen and Tapon 2008). The decreased expression of dBCAS2 by RT-PCR was shown in the upper panel.…”

“…Previous studies have shown that depletion of splicing factors such as dMAFP1 and dPrp38 affects Drosophila cell proliferation and induces apoptosis (Andersen and Tapon 2008). We have found that depletion of BCAS2 resulted in splicing defects and wing deformation.…”

“…1; Table 2). Previous reports have illustrated that depletion of spliceosomal factors, such as ASF and dMFAP1, cause G2/M arrest (Bernstein and Coughlin 1998;Li et al 2005;Andersen and Tapon 2008). Similarly, other members of the hPrp19 complex, hPrp19 and PLRG1, which are both required for RNA splicing, caused lethality at an early stage of embryo development when knocked out in mice (Fortschegger et al 2007;Kleinridders et al 2009).…”

“…To test this hypothesis, RNA extracted from escaper Act5C-GAL4/UAS-dBCAS2 dsRNA third instar larvae was analyzed by quantitative RT-PCR. We chose the transcripts of γ-tubulin and hpo, as reported (Andersen and Tapon 2008), to test whether dBCAS2-knockdown larvae exhibited abnormal pre-mRNA splicing in vivo. We found that the mRNA of γ-tubulin and hpo decreased by 30% to 50%, compared with the wild type, and the premRNA of γ-tubulin and hpo accumulated by 2.7-fold and fourfold, respectively, in dBCAS2-knockdown larvae ( Fig.…”

Mitotic control by mRNA splicing regulators ensures primary cilia formation

“…In the present study, we selected splicing regulators such as SON (Ahn et al 2011) and XAB2 (Andersen & Tapon 2008), which were associated with mitosis regulation and ciliogenesis in the previous study ). Our results from knockdown experiments reveal that these regulators are essential for G2/M transition and mitosis exit of the cells under serum addition, and for cilia assembly of the cells under serum starvation.…”