title: Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.
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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Zhang
authors.forename: Fuxing
authors.initials: F
authors.lastname: Zeng
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Hui
authors.initials: H
authors.lastname: Lv
authors.forename: Liwen
authors.initials: L
authors.lastname: Niu
authors.forename: Maikun
authors.initials: M
authors.lastname: Teng
authors.forename: Xu
authors.initials: X
authors.lastname: Li
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D034664:Heterogeneous-Nuclear Ribonucleoprotein L / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D012313:RNA
keywords: Crystal Structure; Protein Structure; RNA Binding; RNA Looping; RNA Splicing; RNA-Protein Interaction; RNA-binding Proteins; RRM Domain; hnRNP-L