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Context-dependent regulatory mechanism of the splicing factor hnRNP L.

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.

SUBMITTER: Motta-Mena LB

PROVIDER: S-EPMC2818868 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Context-dependent regulatory mechanism of the splicing factor hnRNP L.

Motta-Mena Laura B LB Heyd Florian F Lynch Kristen W KW

Molecular cell 20100101 2

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 ac ...[more]

PMID: 20122404

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Project description:7SK small nuclear RNA (snRNA) is an abundant and ubiquitously expressed noncoding RNA that functions to modulate the activity of RNA Polymerase II (RNAPII) in part by stabilizing distinct pools of 7SK-protein complexes. Prevailing models suggest that the secondary structure of 7SK is dynamically remodeled within its alternative RNA-protein pools such that its architecture differentially regulates the exchange of cognate binding partners. The nuclear hnRNP A1/A2 proteins influence the biology of 7SK snRNA via processes that require an intact stem loop (SL) 3 domain; however, the molecular details by which hnRNPs assemble onto 7SK snRNA are yet to be described. Here, we have taken an integrated approach to present a detailed description of the 7SK-hnRNP A1 complex. We show that unbound 7SK snRNA adopts at least two major conformations in solution, with significant structural differences localizing to the SL2-3 linker and the base of SL3. Phylogenetic analysis indicates that this same region is the least genetically conserved feature of 7SK snRNA. By performing DMS modifications with the presence of excess protein, we reveal that hnRNP A1 binds with selectivity to SL3 through mechanisms that increase the flexibility of the RNA adjacent to putative binding sites. Calorimetric titrations further validate that hnRNP A1-SL3 assembly is complex with the affinity of discrete binding events modulated by the surrounding RNA structure. To interpret this context-dependent binding phenomenon, we determined a 3D model of SL3 to show that it folds to position minimal hnRNP A1/A2 binding sites (5'-Y/RAG-3') within different local environments. SL3-protein complexes resolved by SEC-MALS-SAXS confirm that up to four hnRNP A1 proteins bind along the entire surface of SL3 via interactions that preserve the overall structural integrity of this domain. In sum, the collective results presented here reveal a specific role for a folded SL3 domain to scaffold hnRNP A1/A2-7SK assembly via mechanisms modulated by the surrounding RNA structure.

Dataset Information

Context-dependent regulatory mechanism of the splicing factor hnRNP L.

Publications

Context-dependent regulatory mechanism of the splicing factor hnRNP L.

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