Altered maturation of sequences at the 3′ terminus of 5S gene transcripts in a Saccharomyces cerevisiae mutant that lacks a RNA processing endonuclease.

Abstract

Sequences at the immediate 3′ terminus of several eukaryotic primary transcripts, synthesised just before the termination of transcription, are often lost during RNA processing. The rna82.1 mutation in Saccharomyces cerevisiae appears to result in a deficiency of the endonuclease that removes such sequences from certain yeast transcripts. Some small RNAs of rna82.1 cells are a few nucleotides longer than their counterparts in wild‐type S. cerevisiae. The 5S rRNAs made during very short pulse‐labellings of the mutant have, relative to the mature 121 nucleotide 5S RNA of wild‐type cells, an additional 7, 11 or 13 nucleotides at their 3′ terminus. These 5S forms reveal sites upon 5S genes where transcription probably terminates in vivo. The extra nucleotides upon 5S RNAs in rna82.1 cells are lost very slowly by sequential removal from the 3′ terminus. Through this 3′‐5′ exonuclease action the total 5S RNA of the mutant possesses several 3′‐terminal sequences yet is mostly only 0‐3 nucleotides longer than in wild‐type S. cerevisiae. Just one or two of these 3′‐terminal sequences serve as a substrate in vivo for a poly(A) polymerase since a small proportion of rna82.1 5S RNAs terminate in the sequence: CAAUCUUU(A)n.

Processing of the 3′ sequence extensions upon the 5S rRNA of a mutant yeast in Xenopus laevis germinal vesicle extract

“…The major primary transcripts of the 5s gene in yeast are probably equivalent to the 5s forms of 128 and 132-134 nucleotides synthesised during very short pulse-labelling of rna82.1 cells [9]. RNAs of identical lengths are made by transcription in vitro of Saccharomyces cerevisiae 5s genes (unpublished results).…”

“…RNAs of identical lengths are made by transcription in vitro of Saccharomyces cerevisiae 5s genes (unpublished results). While in RNA82' cells, 3' fragments of the sequence U-U-A-U-U-U-C[U-U-U-U(U-U)] are removed rapidly from these transcripts by the processing endonuclease, giving a 121-nucleotide mature 5s molecule [9], these sequences remain at the 3' end of the pulse-labelled rna82.1 RNA used for the experiment in Fig. 2.…”

“…Saccharomyces cerevisiae pulse-labelled small RNA was prepared from a culture of the endonuclease-deficient (rna82.Z) strain p46 pulsed for 10min at 25°C with [3H]uridine as described in [9]. 5s RNA of 121 -134 nucleotides was purified from this fraction by preparative gel electrophoresis, this RNA being located on the gel by running in an adjacent slot marker 32P-labelled samples of yeast pre-tRNA"" (1 35 nucleotides) and wild-type 5s RNA (121 nucleotides).…”

“…[3H]Uridine-labelled 121-nucleotide 5s RNA of RNA82+ cells was prepared by gel electrophoresis of the total small RNA of a culture of strain S2072D [9] labelled 60min at 25 "C. It was located on the gel by staining. This, together with the 3'-endlabelling of 5s RNA samples by addition of [5'-32P]pCp (catalysed by phage T4 RNA ligase) and the analysis of the 3' termini of these samples, were as described previously [9].…”

“…This, together with the 3'-endlabelling of 5s RNA samples by addition of [5'-32P]pCp (catalysed by phage T4 RNA ligase) and the analysis of the 3' termini of these samples, were as described previously [9].…”