The Solution Structure And Functional Study Of The Human RBM5RRM2&the NMR Study Of The Interaction Between HP1and Borealin

In this thesis, we will present two topics. One focuses on the interaction between RBM5and the RNA presenting in the chapter1, the other on the interaction between HP1α and Borealin presenting in the charpter2.Alternative splicing, in which the different combinations of exons can give rise to the diverse isoforms of the eukaryote genomes. It is known that the proportion of the alternatively spliced multi-exon human genes has expanded to95%. The processes of alternative splicing not only generated the protein isoforms, but also involved in the post-transcriptional regulations.Many of tumor growing events appear to be affected by changes in alternative splicing, especially on the area of apoptosis. It has been reported that a large number of genes involved in apoptosis regulate by alternative splicing. Such as the genes of Fas receptor, Caspase-2and Bcl-x et al. often produce protein isoforms with opposing roles in promoting or preventing cell death. Although the number of alternative splicing evens have been observed in different cancer cells, few demonstrated in mechanisms clearly.Like other levels of gene control, this complicated processing involves both cis-acting elements and trans-acting factors. The cis-acting elements are composed of some short sequences in the pre-mRNA. The trans-acting factors are some cellular RNAs and RNA binding proteins related to the pre-mRNA. Over the past two decades, a number of the complex structures of protein and RNA have been determined both by the X-ray crystallography and NMR spectroscopy that underline the mechanisms of the alternative splicing regulation.RBM5also known as LUCA-15or H37is silenced in70-80%of lung cancers. These observations suggest that both decreased and increased levels of RBM5can play a role in tumor progression. RBM5is a protein of815amino acids with sequence motifs characteristic of RNA-binding factors, include two RRM, two zinc-fingers, one RS, an OCtamer REpeat (OCRE) domain and one glycine patch. Recently, the biochemical functions of RBM5were demonstrated in regulation of Fas and caspase-2alternative splicing. For caspase-2pre-mRNA, Fushimi and his colleges identified a cis-acting element on inton9, which can be directly recognized by RBM5. In the present study, we solved the solution structure of RBM5RRM2, which revealed a canonic RRM domain with4beta strand formed a plant for RNA binding. Using the Fluorescent Polarization experiment, we found that the RRM2domain showed a slight preference to the5’-CUCUUC-3’oligonucleotide derived from the caspase-2intron sequence. Then the chemical shift perturbation analyses revealed the RBM5RRM2directly contacts with the RNA through its β-sheet surface and the C-terminal extension is important for RNA binding. To further confirm that these most perturbed residues involved in RNA binding, we performed three point mutations experiments for their ability to bind the5’-CUCUUC-3’RNA. To investigate the function of the RBM5RRM2C-terminal extension, we measured the binding affinities of the CUCUUC RNA interaction with the different protein fragments by fluorescence polarization assay.This work further defined the interaction between RBM5and caspase-2pre-mRNA and provided a molecular basis for the mechanism of RBM5biological function in caspase-2alternative splicing regulation.In chapter2, we used NMR spectroscopy to monitor the chemical shift perturbation upon Borealin peptide (223-240) binding to HP1α CSD. With cooperation of Professor Yao’s lab, we proved that Borealin-HP1interaction exists and may participate in targeting CPC to centromere since the Borealin mutant of PxVxL motif lost its ability to localizing to centromere.