期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 31, 页码 22636-22649出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.463901
关键词
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资金
- Chinese Ministry of Science and Technology [2012CB917200, 2009CB825500]
- Chinese National Natural Science Foundation [31270014, 31130018, 30900224]
- Science and Technological Fund of Anhui Province for Outstanding Youth Grant [10040606Y11]
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 angstrom, respectively. These RRMs all adopt the typical beta 1 alpha 1 beta 2 beta 3 alpha 2 beta 4 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.
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