Abstract
Based on a correlation between expression patterns of an abundant tissue-specific small nuclear ribonucleoprotein (N protein) and the calcitonin gene-related protein (CGRP) splicing choice, a small nuclear ribonucleoprotein (the N factor) has been hypothesized to potentially function as a trans-acting factor involved in the regulation of the alternative splicing of the calcitonin/CGRP transcript. RNA analysis indicated that most rat, human, and simian cell lines and tissues making the CGRP mRNA splicing choice expressed the N factor mRNA. These data led us to address the effect of ectopic expression of the N factor in HeLa cells, which exhibit a calcitonin splicing choice when expressing the calcitonin/CGRP gene. Expression of the N factor exerts no effect on the calcitonin/CGRP splicing choice in HeLa cells. Furthermore, several cell lines such as the human 293 cell line make the CGRP mRNA splicing choice in the absence of any detectable level of the mRNA encoding the N factor. Together, these data reveal that the N protein is neither sufficient nor required for the tissue-specific CGRP splicing decision and that the N protein is not the trans-acting factor regulating the alternative splicing of the calcitonin/CGRP gene.
Highlights
Based on a correlation between expression patterns pressed primarily in mammalian neural and cardiac tissues of an abundant tissue-specific small nuclear ribonucle(-4)
Because we have shown that N factor mRNA is detected either only at a very low level or not at all in certain Rat-1 F4 clones, which make the calcitonin gene-related protein (CGRP) splicingchoice, and is undetectable in the human 293 cell, we investigated the expression of the Nfactorin cell types making either calcitonin or CGRP splicing choices by the most sensitive technology available: polymerase chain reaction
Closely related to CV-1 cells but expressing the SV40 large T antigen, have been shown to allow the alternative splicing of the L1 gene of adenovirus (26), which does not occur in CV-1 cells (25)
Summary
Fetal bovine serum, methotrexate a t a final concentration of 0.6 mM, Analysis of N Factor Expression inRat Cell Lines and and 0.6 mg/ml G418 (12). RNase digestion was performed by adding 350 pl of buffer (10 mM Tris-C1, pH 7.5, liver and in the normal thyroid (THY)RNA, whereas it was present at a high level in medullary thyroid carcinoma RNA ( WF). The Calcitonin/CGRPExpression and Its Splicing Patternmembrane was washed at high stringency Analysis of RNAs from normal thyroid tissue (Fig. 5B, THY) and medullary thyroidcarcinoma (Fig. 2B, W F ) or from cells pletion in the presence of yeast tRNAs (C). C, 10 pg of total RNAs from B50 and CA77 rat cell lines were analyzed as described above. Derived from these tumors (CA77) or of neural origin (B50, B103) (Fig. 2C) revealed the expression of the endogenous calcitonin/CGRP gene
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