Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.
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Zhang W, Zeng F, Liu Y, Zhao Y, Lv H, Niu L, Teng M, Li X
Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.
J Biol Chem. 2013 Aug 2;288(31):22636-49. doi: 10.1074/jbc.M113.463901. Epub 2013 Jun 19.
- PubMed ID
- 23782695 [ View in PubMed]
- 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 A, respectively. These RRMs all adopt the typical beta1alpha1beta2beta3alpha2beta4 topology, except for an unusual fifth beta-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two beta-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the beta-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.