Download Investigating the Function of Small Nuclear Ribonucleoprotein

Investigating the Function of Small Nuclear Ribonucleoprotein
Polypeptide N (snRPN) in Alternative Spliclng
Adviser:Chin Li
Presenter:Chiung-Chun Shen
Alternative splicing is a key molecular event that allows for production of
multiple mature mRNA isoforms. Through this process, the coding capacity of a
single gene is increased by expressing several related proteins with diverse and even
antagonistic functions. Aberration of alternative splicing affects essential biologic
processes and probably leads to pathogenesis, including cancer. Mutations in cisacting splicing elements and changes in the expression or activity of regulatory
proteins will both lead to abnormal splicing. In previous study, we discovered not
only the aberrant alternative splicing events but also up-regulated expression of
specific splicing factors in transitional cell carcinoma cell lines. One of the upregulated splicing factors is small nuclear ribonucleoprotein polypeptide N (snRPN).
snRPN is a well-known imprinted gene frequently with its methylation status altered
in the Prader-Willi syndrome. It has been shown that snRPN is first incorporated into
the U2 snRNP and then into U1 snRNP when expressed at higher level. Thus far, the
function of snRPN in alternative splicing is poorly investigated. Hence, the main
objective of this study is to clarify the function of snRPN in alternative splicing.
First, recombinant snRPN protein was purified and used to generate the
customized antibody. Meantimes, we also create a HeLa cell line that constitutively
expresses snRPN. The specificity and efficiency of the polyclonal anti-snRPN
antibody was confirmed by By western blotting and immunofluorescence staining.
Specifically, the cellular location of snRPN is in the nucleus, consistent to its function
in splicing. In order to investigate the function of snRPN in alternative splicing, the
transcriptome of snRPN-expressing HeLa cell line was analyzed by Affymetrix
GeneChip exon ST 1.0 array using HeLa cells as reference. Among the genes
identified to likely exhibit alternative splicing, thirty genes were selected and the
aberrant splicing event was examined by RT-PCR. However, only few altered splicing
events can be confirmed. Finally, gelsolin, which shows most significant and clear
alternative splicing, was selected for further study. The aberrant splicing events of
Gelsolin include partial intron 14 inclusion (alternative 5’ splice site selection),
exclusion of N terminal sequence of exon 15 (alternative 3’ splice site selection), and
complete exon 16 exclusion. By employing quantitative reverse transcriptase-PCR,
we confirmed that expression of snRPN in HeLa significantly reduces the frequency
of partial intron 14 inclusion. We also found that snRPN is incorporated into U2
snRNP by a linear 10-50% glycerol gradient. Taken together, our data demonstrates
that incorporating of snRPN into snRNPs leads to altered splice site selection.