The Structural And Functional Research Of An Alternative Splicing Regulator HnRNP L And A Subunit Of A Histone Chaperone FACT Complex SSRP1

Part I:Pre-mRNA splicing is a crucial mechanisom for gene expression. It is regulated at different levels in a tissue-or developmental stage-specific manner. Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. The overall function of alternative splicing is to increase the diversity of mRNA expressed from the genome, generating different splicing isoforms from one pre-mRNA to fulfill various functions. The aberrant regulation of alternative splicing leads to human diseases. hnRNP L is an important splicing regulator involved in alternative-splicing regulation of many genes. Besides, it plays roles in chromatin modification, transcriptional regulation, mRNA export of intronless genes, IRES-dependent translation and mRNA stability regulation.hnRNP L contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In our study, results of surface plasmon resonance (SPR) spectroscopy assays revealed that all the four RRM domains contribute to RNA-binding. Compared to RRM12, RRM34shoulders the major responsibility for RNA binding of hnRNP-L. Then, we elucidated the crystal structures of RRM1and RRM34of hnRNP-L at2.0A and1.8A, respectively. These RRMs all adopt the typical β1α1β2β3α2β4topology except the presence of an unusual fifth P-strand in RRM3. RRM3and RRM4interact intimately with each other mainly through helical surfaces, leading the two β-sheets to point to the opposite directions. Structure-based mutations and SPR assays results suggested that all the antiparallel β-sheets of RRMs are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays (ss-FRET), together with cross-linking and dynamic light scattering (DLS) assays, demonstrated that hnRNP L RRM34facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and ITC 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 either as an activator or repressor. Our results also enlighten the possible ability of hnRNP L in recruiting additional factors through RNA looping without direct protein-protein interactions. Part II:Chromatin is a kind of densely packed and tightly regulated nucleoprotein complex that stores the cellular genetic material in a stable yet readily accessible form. To overcome the inhibitory effects of nucleosomes on the accessibility of DNA during basic chromatin-templated progresses such as transcription, DNA replication and repair, it needs assistance from many factors to alter the chromatin structure. FACT (FAcilitates Chromatin Transcription) complex is an important histone chaperone, which reorganizes nucleosome without hydrolyzing ATP and translocating histone octamers relative to DNA. It can not only disrupt core histone-histone and histone-DNA interactions, but also possess the ability to deposit H2A-H2B dimer and (H3-H4)2tetramer onto DNA. Human FACT complex is composed of Spt16and SSRP1. SSRP1protein contains three well-defined domains:the N-terminal/dimerization Domain (NTD/DD), middle domain (MD), and HMG-1domain (HMG).We determined the crystal structure of the middle domain of SSRP1(SSRP1-M) at a resolution of1.93A. The SSRP1-M structure adopts a compact double PH domain architecture. In structural comparison to typical PH domains, PHI domain contains extra two antiparallel strands linked by a helix, which are inserted between the last strand and the C-terminal helix. The residues constituting such unique super secondary structure are conserved through different species. The analysis of electrostatic potential surface of the structure of SSRP1-M showed that one positively charged ridge region exists on each side of surface, suggesting its DNA binding ability. The results of EMSA assays and mutagenesis assays revealed that SSRP1-M binds nonspecificly with DNA and the two positively charged regions are involved in DNA binding. Besides, our pull-down experiments showed that SSRP1-M doesn’t interact with histone, differently from its homologous protein in yeast Pob3-M. Our study provides structural and biochemical insights for SSRP1’s detailed roles in the reorganization and reassembly of nucleosomes.