Context-dependent regulatory mechanism of the splicing factor hnRNP L - PubMed (original) (raw)

Context-dependent regulatory mechanism of the splicing factor hnRNP L

Laura B Motta-Mena et al. Mol Cell. 2010.

Abstract

Splicing regulatory proteins often have distinct activities when bound to exons versus introns. However, less clear is whether variables aside from location can influence activity. HnRNP L binds to a motif present in both CD45 variable exons 4 and 5 to affect their coordinate repression. Here, we show that, in contrast to its direct repression of exon 4, hnRNP L represses exon 5 by countering the activity of a neighboring splicing enhancer. In the absence of the enhancer, hnRNP L unexpectedly activates exon inclusion. As the splice sites flanking exon 4 and 5 are distinct, we directly examined the effect of varying splice site strength on the mechanism of hnRNP L function. Remarkably, binding of hnRNP L to an exon represses strong splice sites but enhances weak splice sites. A model in which hnRNP L stabilizes snRNP binding can explain both effects in a manner determined by the inherent snRNP-substrate affinity.

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Figures

Figure 1

Figure 1. Differential arrangement of the ARS regulatory element in the three variable exons (4, 5, and 6) of the CD45 gene

(A) Schematic of the human CD45 gene and its three variable exons. Exons and introns are represented by boxes or lines, respectively. The ARS-containing element (darker grey square) is embedded within a single region in exons 4 and 6, however in exon 5 the ARS is divided into two regions by an exonic splicing enhancer (ESE) (black square). The ARS consensus sequence is shown below. (B) Sequence of the regions important for regulation of exon 5: the two ARS-containing sequences, labeled S1 and S2, and the ESE. The ARS-core motif is underlined in both the S1 and S2 elements. (C) Comparison of intronic sequence flanking exons 4 and 5 with polypyrimidine tract underlined.

Figure 2

Figure 2. HnRNP L binds to the ARS core of exons 4 and 5 with different co-associated proteins

(A) Top, silver stain of RNA-affinity pulldowns done with exon 4 (ESS1) and exon 5 (WT, ΔESE, ΔS1S2) probes. Asterisk indicates hnRNP L, PTB, and hnRNP E2. Bottom, Western blot analysis of the same RNA-pulldown samples using antibodies against previously characterized ESS1-binding proteins. (B) RNA mobility-shift experiments of radiolabeled versions of the probes from panel A, incubated with increasing amounts of recombinant hnRNP L (top) or hnRNP LL (bottom) proteins. (C) RT-PCR of in vitro splicing reactions in resting JSL1 nuclear extract supplemented with recombinant hnRNP L (left panel) or hnRNP LL (right panel). Schematics of the minigenes used in these experiments are shown at the top. Hatched boxes correspond to substitution mutation of regulatory sequences. (D) RT-PCR of in vitro splicing reactions of CD5-derived RNA incubated in JSL1 nuclear extract supplemented with Flag-tagged PSF protein purified from resting (R) or stimulated (S) JSL1 cells. Western blot with anti-Flag antibody of protein fractions added to the reactions above.

Figure 3

Figure 3. SF2/ASF is a candidate ESE-binding protein of CD45 exon 5

(A) Top, RNA-mobility shift experiment using radiolabeled E5-WT and ESE RNAs and recombinant SF2/ASF. Bottom, mobility shift assay done with indicated RNAs in JSL1 nuclear extract, in the absence (−) or presence (+) of anti-SF2/ASF (α-SF2/ASF) or anti-9G8 (α-9G8) antibody. Super-shifted complexes are indicated with asterisk. (B) Western blot with anti-SF2/ASF of RNA-affinity pulldowns done with nonspecific (NS) and exon 5 (E5, ΔESE, ΔS1S2) probes as in Figure 2A. (C) RT-PCR of in vitro splicing reactions done with indicated RNAs in JSL1 nuclear extract supplemented with recombinant SF2/ASF (top) or 9G8 (bottom). The numbers shown below each panel represent the mean exon inclusion, n=3.

Figure 4

Figure 4. The ARS motifs in exon 5 repress the exon by antagonizing the activity of the ESE

(A) RT-PCR analysis of RNA derived from resting (−PMA) or stimulated (+PMA) JSL1 clones that stably express WT (SC5) and mutant (ΔS1S2, ΔESE, ΔESE+S1S2) exon 5 minigenes, schematics of which are shown at the top. White boxes and black lines correspond to sequence from the human β-globin gene. Rest of coloration is consistent with Figures 1 and 2. Bottom, mean percent inclusion of exon 5 +/− SD, n>6. (B) RT-PCR of in vitro splicing reactions using WT CD5 substrate in the absence (−) or presence of increasing amounts of various exogenous RNA competitors. Mean % inclusion is shown below, n>3. (C) Left, UV crosslinking of radiolabeled exon 5 probes (WT and ESE) with JSL1 nuclear extract or recombinant proteins as indicated.

Figure 5

Figure 5. The ESE in exon 5 activates the formation of A-complex on its upstream intron

(A) RT-PCR of in vitro splicing reactions. Schematics of each of the minigenes used are shown on the left. Graph represents mean +/− SD from 3 independent experiments. (B) Radiolabeled RNA substrates derived from each of the minigenes shown in panel A were incubated in nuclear extract for the times indicated and the resulting spliceosome complexes were resolved on native agarose gels. (C) Assembly and RT-PCR analysis done in the absence (−) or presence (+) of 100 ng of recombinant hnRNP L protein. (D) Same as panel C except with ΔS1S2 substrate (E) Same as in panel C, except reactions were incubated in the absence (−) or presence (+) of 10 pmol of CA-oligo.

Figure 6

Figure 6. HnRNP L represses strong splice sites but activates weak splice sites

(A) Mean exon inclusion +/− SD from RT-PCR of stable cell lines expressing the minigenes shown, done in triplicate. Black boxes and bold black lines represent exonic and intronic sequence from CD45 exon 4 respectively, rest of coloration is consistent with other figures. Glo-weak and glo-weak S1S2 minigenes carry mutations in the 5’ss downstream of the central exon. (B) Mean exon inclusion +/− SD from triplicate in vitro splicing reactions, done in the absence or presence of MS2-hRNP L, using RNAs transcribed from minigenes shown. Numbers shown for 5’ss represent score for 5′ splice site strength (

http://genes.mit.edu/burgelab/maxent/Xmaxentscan\_scoreseq.html

).

Figure 7

Figure 7. Model for hnRNP L function

(A) Interaction of hnRNP L with U1 and U2 snRNPs bound to strong flanking splice sites sequesters them in an inactive conformation that cannot progress further in the spliceosome assembly pathway. (B) However, if an ARS-containing exon is flanked by weak splice sites, then the interaction between U1 and U2 snRNPs and the exon-intron boundary is highly inefficient. In such a case interaction of hnRNP L with U1 and U2 may stabilize their interaction with the splice sites thus promoting progression through assembly pathway. (C) If the hnRNP L-binding sites are located within an intron then the interaction of U1 and/or U2 with hnRNP L would be predicted to bring these snRNPs together in a productive complex.

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