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. 2005 Jul 20;24(14):2646-55.
doi: 10.1038/sj.emboj.7600723. Epub 2005 Jun 16.

Negative feedback regulation among SR splicing factors encoded by Rbp1 and Rbp1-like in Drosophila

Supriya Kumar  1 , A Javier Lopez
Affiliations

Negative feedback regulation among SR splicing factors encoded by Rbp1 and Rbp1-like in Drosophila

Supriya Kumar et al. EMBO J. .

Abstract

SR proteins constitute a widely conserved family of splicing regulators. Negative autoregulation of SR proteins has been proposed to exert homeostatic control on the splicing environment, but few examples have been studied and the role of isoforms that lack the RS domain is unclear. We show that genes Rbp1 and Rbp1-like, which encode Drosophila homologs of mammalian SRp20, negatively autoregulate and crossregulate at the level of alternative 3' splice site selection. This adjusts the relative expression of isoforms with either an RS domain or unrelated C-terminal domains (ALT) that are rich in serine and threonine. The effects of RBP1-ALT on splicing of doublesex and Rbp1-like are opposite to those of RBP1-RS and RBP1L-RS. RBP1-ALT and -RS exert opposing negative feedback on the ALT/RS ratio. However, RBP1-ALT inhibits the expression of RBP1-RS while stimulating that of RBP1L-RS. This asymmetry may contribute to changes in the RBP1-RS/RBP1L-RS ratio that are observed during development. These results provide the first example of a feedback-regulated SR protein network with evidence of an active homeostatic role for alternative isoforms.

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Figures

Figure 1
Figure 1
Effects of RBP1-RS, RBP1L-RS and 9G8 on splicing of dsx RNA. (A) Gene structure and alternative splicing patterns of dsx. F: female 3′ splice site; M: male 3′ splice site; C: novel 3′ splice site. Arrows show the locations of PCR primers. (B) Immunoblot analysis of RBP1 and RBP1L protein expression in control and overexpressing cells. (C) RT–PCR analysis of endogenous dsx RNAs in SL2 cells overexpressing RBP1-RS, RBP1L-RS or 9G8. rp49 is a coamplified internal standard. The graph shows levels of alternative dsx mRNAs relative to rp49 in control and experimental cells. The average and standard error for four experiments are shown in each case. *P<0.05; **P<0.01. (D) RT–PCR analysis of endogenous tra and tra2 RNAs in SL2 cells overexpressing RBP1-RS, RBP1L-RS or 9G8.
Figure 2
Figure 2
Levels of Rbp1-RS and Rbp1L-RS mRNAs are unaffected by a 50% reduction of gene dosage. The upper left panel shows RT–PCR analysis of mRNA in adult females heterozygous for Df(1)C246 or Df(3R)cu. The ratios of Rbp1-RS or Rbp1L-RS to rp49 are plotted in the right panel, showing the average of three replicates and standard error. The lower left panel shows that PCR can distinguish a 50% reduction of input cDNA for either mRNA.
Figure 3
Figure 3
Feedback and crossregulation of Rbp1 and Rbp1-like. (A, upper and lower panels) RT–PCR analysis of endogenous mRNAs for SR proteins in SL2 cells overexpressing RBP1-RS, RBP1L-RS or 9G8. (B) RT–PCR analysis of mRNA levels following RNAi-mediated knockdown of Rbp1 or Rbp1-like in SL2 cells. (C) Immunoblot analysis of RBP1-RS and RBP1L-RS after RNAi in SL2 cells. The lower panel shows γ-tubulin as a loading control. (D) RT–PCR analysis of mRNA levels following RNAi knockdown of Rbp1 or Rbp1-like in embryos. (E) Immunoblot of RBP1-RS and RBP1L-RS in normal Drosophila embryos and adults. Graphs in panels B and D show the ratios of Rbp1-RS and Rbp1L-RS to rp49 mRNA, plotting the mean for three experiments and standard errors. *P<0.1; **P<0.05; ***P<0.01.
Figure 4
Figure 4
Alternative splicing of Rbp1 and Rbp1-like transcripts. (A) Diagrams of Rbp1 and Rbp1-like showing relative locations of alternative 3′ splice sites, start and stop codons (not to scale). The coding regions for the RBD, RS and ALT domains are labeled. Arrows show the relative positions of PCR primers used to detect the endogenous mRNAs. (B) Amino-acid sequences of the RS and ALT domains of RBP1 and RBP1L. The putative PEST domain of RBP1L-ALT is shaded. (C) RT–PCR analysis of endogenous Rbp1 and Rbp1L isoforms in control SL2 cells; Rbp1 mRNAs were amplified with a single primer set for 27 cycles; Rbp1L-RS and Rp1L-Alt were amplified with separate primer sets for 30 cycles. (D) RT–PCR analysis of endogenous RS and ALT isoforms in SL2 cells that overexpress RBP1-RS or RBP1L-RS. The endogenous ALT and RS isoforms were amplified with separate primer sets in both cases; amplification and loading were adjusted to obtain comparable signals for all isoforms from control cells.
Figure 5
Figure 5
Lariat analysis of 3′ splice site selection. (A) Diagram of relevant alternative splicing events in Rbp1 and Rbp1-like. Arrows show relative position and orientation of primers for lariat analysis. (B) Strategy used for RT–PCR analysis of lariats. In the cDNA product, the F and B priming sites become convergent across the branch junction (bp). (C) RT–PCR analysis of Rbp1 and Rbp1-like lariats in SL2 cells after knockdown of debranching enzyme expression.
Figure 6
Figure 6
Functional analysis of the RBP1-ALT protein. (A) Structures of RBP1-RS, RBP1-ALT and mutant RBP1-delRS. (B) Immunoblot analysis of SL2 cells overexpressing RBP1-ALT or RBP1-delRS. The antibody was raised against the RRM exon and detects both proteins as well as RBP1-RS and RBP1L-RS. The blot was also probed with an antibody against hrp48 as a quantitative standard. The bands corresponding to RBP1-RS and RBP1L-RS were identified by overexpression of the corresponding cDNAs (Figure 1B) and knockdown of the mRNAs (Figure 3C). (C) RT–PCR analysis of dsx, tra and tra2 mRNA isoforms in SL2 cells overexpressing RBP1-ALT or RBP1-delRS. (D) RT–PCR analysis of Rbp1 and Rbp1-like mRNAs in cells overexpressing RBP1-ALT or RBP1-delRS. The endogenous RS and ALT isoforms of both genes were amplified with separate primers; cycle number and loading were adjusted to equalize the signals from control cells. (E) RT–PCR analysis of pre-mRNA levels for Rbp1 and dsx. Each experiment of panels B–E was replicated at least three times and similar results were obtained.
Figure 7
Figure 7
Regulatory circuit showing homeostatic regulation of Rbp1 and Rbp1-like. Horizontal arrows indicate alternative splicing patterns. Curved arrows indicate stimulation and bars indicate inhibition. Effects on tra and tra2 splicing are not shown (see text).
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