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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Authors: | Wenjuan Zhang Fuxing Zeng Yiwei Liu Yan Zhao Hui Lv Liwen Niu Maikun Teng Xu Li |
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Institution: | From the ‡Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China and ;the §Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, China |
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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. |
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Keywords: | Crystal Structure Protein Structure RNA Splicing RNA-binding Proteins RNA-Protein Interaction RNA Binding RNA Looping RRM Domain hnRNP-L |
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