Horn, David Andrew; (1993) SmN: Expression and potential role in alternative mRNA splicing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Sm proteins associate with small nuclear RNA (snRNA) molecules to form small nuclear ribonucleoprotein particles (snRNPs) which are essential for pre mRNA splicing. A recently identified Sm protein, SmN is expressed in a tissue specific manner and is closely related to the constitutively expressed SmB protein. SmN is abundant in neurons and is expressed at lower levels in cardiac muscle while being undetectable in other tissues; it is therefore the first example of a mammalian snRNP protein of this kind. Human, rat and mouse forms of the protein are 100% identical. Mutation at the SmN locus has also been implicated in Prader-Willi syndrome suggesting an important function for this protein. Expression of SmN has been characterised in a number of neuronal and non-neuronal cell lines, in EC cells during differentiation and in tissues during embryonic development using the KSm5 antibody in conjunction with Western blotting. The KSm5 antibody has also been used to examine Sm protein distribution by immunofiourescence, and RNA probes have been used to examine SmN and SmB mRNA expression and SmN mRNA distribution in the mouse brain. The distribution of the closely related SmN, B and B' proteins has also been defined in human tissues. SmN distribution, and its association with the splicing machinery has raised the possibility that it is involved in alternative splicing. In particular, cells and tissues which express SmN have been shown to have the ability to follow an alternative splicing pathway resulting in the production of the CGRP mRNA from the primary transcript of the CALC-I gene. Characterisation of SmN expression in a number of cells and tissues allowed an investigation into the possible role of SmN in alternative splicing. A number of cell lines ectopically expressing SmN were also constructed in order to examine the role of SmN. Using a number of the cell lines and tissues characterised above, as well as the stable cell lines, the putatative role of SmN in alternatively splicing has been examined. Using quantitative PCR assays it appears that SmN is neither necessary nor sufficient to guide alternative splicing of a number of mRNAs which appeared to be candidates for regulation by SmN, from the literature, including CALC-I, NCAM (VASE exon) and c-src. A pair of CALC-I gene constructs have also been used to identify a cis-acting sequence not originally thought to be involved in CALC-I alternative splicing. The data presented here and other recent results have been used to speculate on other possible roles for SmN. The SmN expressing stable cell lines provide a model in which to examine SmN functions further and the ND neuronal cell lines provide a good model to examine CALC-I alternative splicing.

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