Abstract
Publisher Summary The detection, purification, and partial characterization of 5′-exoribonuclease 1 (Xrn1) from Saccharomyces cerevisiae as a 160-kDa RNase was reported in 1980 and 1985. The enzyme is a processive exonuclease hydrolyzing RNA from the 5′ end with the production of 5′-mononucleofides. The XRN1 gene was cloned in several laboratories, and the results showed that it encodes a 175-kDa protein. This chapter describes the purification of Xrn1 from yeast cells containing the gene in a GALI0 expression vector. Assay methods for analysis of 5′-exoribonuclease activity as well as the substrate specificity of Xrn1 are also described. The first evidence of the involvement of Xrn1 in RNA metabolism was the finding that yeast cells with a disrupted gene accumulate an internal transcribed spacer fragment of pre-rRNA. It was then found that short-lived mRNAs lacking both the poly(A) tail and the cap structure also accumulate. The findings suggested that Xrn1 is involved in cytoplasmic RNA metabolism and mRNA is hydrolyzed after its deadenylation and decapping.
Published Version
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