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Identification of the Sm N protein and studies on its expression

Sharpe, Neil Grey; (1991) Identification of the Sm N protein and studies on its expression. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The major species of mammalian Sm proteins associate with snRNA molecules to form small nuclear ribonucleoprotein particles (snRNPs) which are essential for pre-mRNA splicing. A series of immunoblots were probed with an anti-Sm monoclonal antibody. These experiments led to the identification of a cell-specific 28kDa protein called Sm N. The expression of the Sm N protein is restricted to cell lines and tissues which have the ability to utilise the alternative splicing pathway of the calcitonin/CGRP gene. This correlation suggests that the Sm N protein may play a role in determining the use of this alternative splicing pathway. A Sm N cDNA clone was isolated by immunoscreening a HeLa λgt11 expression library. Characterisation of the cDNA clone showed that the Sm N protein consists of 240 amino acids. It has a proline-rich carboxyl terminus and it is closely related to the Sm B and B' proteins. Northern blot analysis revealed that the Sm N protein is encoded by a 1.6kb mRNA transcript. RNA analysis showed that the Sm N gene is differentially expressed in HeLa cell lines. The levels of the Sm N protein and mRNA were shown to decline during the differentiation of embryonal carcinoma stem cells to parietal endoderm-like cells. Furthermore, the levels of the Sm N protein decline during the differentiation of embryonal stem cells suggesting that this effect may occur in vivo. The sera of some patients with the autoimmune disease systemic lupus erythematosus (SLE) contain anti-Sm autoantibodies. The anti-Sm monoclonal antibody which was used to identify Sm N recognises a disease autoepitope. In order to localise this epitope on the Sm N protein, a short, 135 nucleotide Sm N clone was obtained by immunoscreening a λgt11 expression library. A lysogen of this clone was used to detect the presence of anti-Sm antibodies in SLE sera. It was found that both anti-Sm and anti-RNP autoantibodies reacted with the fusion protein. This effect was due to amino acid sequence similarities between Sm N and the U1 snRNP-specific C protein. Immunoblotting analysis was used to show that the highly immunoreactive Sm B, B', and D proteins increase in abundance in Vero cells infected with herpes simplex virus type 2. This effect may increase the antigenicity of these Sm proteins.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Identification of the Sm N protein and studies on its expression
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Biological sciences; Pre-mRNA splicing
URI: https://discovery.ucl.ac.uk/id/eprint/10120656
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