Abstract
Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.
Highlights
HnRNP L is a multifunctional RNA-binding protein implicated in alternative splicing regulation, etc
Through structurebased, optimized sequence alignments between Heterogeneous nuclear ribonucleoprotein L (hnRNP L) and PTB, we found that for both RRM1 and RRM34, the residues corresponding to those participating in recognition of C3 in PTB are conserved in Heterogeneous nuclear ribonucleoproteins (hnRNPs) L, whereas those involved in recognition of U2 and U4 are less conserved in hnRNP L (Fig. 4, A and B)
RNA Looping Induced by hnRNP L RRM34—Because we found that the two -sheets of RRM3 and RRM4 align in an antiparallel fashion, facing opposite directions, and that both are involved in RNA binding, which would require the RNA backbone to bend to interact with both RNA recognition motifs (RRMs) domains, we speculated that hnRNP L RRM34 may bind to two separate binding sites within the same RNA by inducing RNA looping
Summary
Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. HnRNP L is an abundant and multifunctional protein that shuttles between the nucleus and cytoplasm It mediates selective exon inclusion during alternative splicing [3], facilitates polyadenylation [4], assists export of intronless gene mRNA [5], promotes internal ribosomal entry site-dependent translation [6, 7], and regulates mRNA stability [8, 9]. We propose models for how hnRNP L functions as an alternative splicing regulator with dual roles and suggest the possibility of hnRNP L-mediated recruitment of other factors without direct interactions
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