the addition of a digestion with an appropriate restriction enzyme which specifically acts against complementary sequences

Considering novel poly isolated mRNAs is whether they are protein coding or not. Human RNase k is a previously established protein belonging to a family conserved in all metazoans. The high conservation of all members along with the fact that the human enzyme is widely expressed in all tissues and developmental stages suggest a very important biological function that is currently under investigation. Human RNase k is represented by a single copy in the human genome and retains the pattern of gene organization that is preserved by almost all known members of the gene family, consisting of three exons interrupted by two introns. The human representative exhibits in vitro endoribonucleolytic activity cleaving preferentially ApG and ApU phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Reducing reagents and site-directed mutagenesis experiments showed that a disulfide bond between cysteine residues 6 and 69 is essential for the ribonucleolytic activity of the enzyme. The present work reports a human subtle alternative splicing event giving rise to a protein coding mRNA variant that lacks 4 nucleotides compared to the previously reported RNase k mRNA isoform. Cloning and expression analysis of this subtly alternatively spliced mRNA was achieved by the development of a modified hybrid selection approach which may provide a tool for the study of similar cases. RNase k-02 mRNA encodes the synthesis of an alternative protein isoform in human cell extracts which follows a cytoplasmic distribution. Subtle alternative splicing events are being observed in an everrising number of human genes, as depicted by the exponential accumulation of EST and high throughput RNA sequencing data. The impact of subtle alternative splicing on protein products depends on whether they are frame-shifting or frame-preserving with D denoting the sequence length between the tandem splice sites. Frame-preserving tandem sites result in the addition or absence of only a few aminoacids, leading to the production of similar protein isoforms which may bear different functional properties. For instance, a D3 subtle alternative splicing event occurring in ATN1 human gene product determines the cellular topology of the corresponding protein isoforms. At the donor site, alternative splicing events in which the distance between two splice sites is 4 nucleotides long are the most frequent. However, their frameshift effect often creates mRNA isoforms that are predicted as nonsense mediated decay targets and therefore these cases have not been systematically investigated. The methodological approach presented allowed us to achieve the cloning of a human RNase k mRNA variant that occurs after a D4 splicing event. This experimental strategy offers several advantages for the study of transcripts generated by alternative splicing compared to conventional RT-PCR approaches.

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