Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast.

Abstract

The temperature-sensitive mutation rna2 causes the accumulation of higher molecular weight transcripts from the ribosomal protein 51 (rp5l) gene of yeast and many other yeast ribosomal protein genes. We have determined the DNA sequence of the rp5l gene, confirming that it contains an intron and that the higher molecular weight transcript is an intron-containing precursor RNA. These data and other experiments suggest that the rna2 mutation affects mRNA processing (splicing) and that the presence of an intron is sufficient to render expression of a gene sensitive to the rna2 mutation. To test these hypotheses, we have inserted the rpSl intron into the coding region of a hybrid Escherichia coli 13-galactosidase gene, thereby interrupting the open reading frame subsequent to the initiating methionine codon. De-spite the presence of the intron, the f3-galactosidase gene is expressed in yeast. Thus, the rp5l intron is properly excised from the normally intronless gene. The presence of the rpSl intron causes the 1B-galactosidase activity to be sensitive to the rna2 mutation, consistent with the notion that this mutation affects gene expression at the level of splicing. The experiments suggest that an intron-containing /8-galactosidase gene can be used in a general way to study mRNA splicing.The temperature-sensitive rna2 mutation of Saccharomyces cerevisiae causes a dramatic decline in the synthesis of most ribosomal proteins when cells carrying the rna2 mutation are shifted to the restrictive temperature. This effect appears to be quite specific for ribosomal protein synthesis-i.e., the rate of synthesis of total protein and of most nonribosomal proteins is relatively unaffected (1). After a shift to the restrictive temperature, mRNA isolated from cells that carry the rna2 mutation is specifically depleted of most ribosomal protein mRNAs, suggesting that the decline in ribosomal protein synthesis is due to a decline in ribosomal protein mRNA levels (2). For many ribosomal proteins, the decrease in mature mRNA levels in cells carrying the rna2 mutation is associated with increased levels of higher molecular weight transcripts (3)(4)(5). In the case of ribosomal protein 51 (rp5l), the mature mRNA and the higher molecular weight transcript were shown by nuclease S1 mapping to be spliced and unspliced transcripts, respectively (3).These data suggest that many yeast ribosomal protein genes contain introns and that the rna2 mutation interferes with ribosomal protein mRNA processing. Here we present the rp51 DNA sequence and identify the rpSl intron. We also demonstrate that the rp51 intron, when inserted into a yeast CYCJlacZ fusion gene (6), is correctly spliced from the fusion transcript, thereby allowing 03-galactosidase expression. Splicing of the rp5l intron from the CYCl-lacZ fusion transcript is further shown to be defective in cells carrying the rna2 mutation when shifted to the nonpermissive temperature. MATERIALS AND METHODSStrains. The haploid S. cerevisiae strain RY26 (a, rna2, ura3-1, ura3-2, adel, ade...

Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes.

“…7B, lane 1. Only the RP51A products are easily visible (28). The RP51B products are very faint, almost certainly a reflection of the lower abundance of these transcripts, the mismatch with the primer, and an even more heterogeneous set of 5' ends.…”

“…This RP51 gene (Fig. 1A) has been subcloned and sequenced (28). The results of these analyses indicate that this RP5J gene codes for a basic protein of 135 amino acids.…”

Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: Intron length effects and activity of a precursor snoRNA

“…Plasmids pSO4 and pSO5 were constructed as follows+ Oligonucleotides JB910 and JB911 were used to amplify the MATa trp1⌬63 his3⌬200 leu2⌬1 ura3-167 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 YSO50 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬dbr1::HIS3 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬dbr1::HIS3 YSO53 MATa trp1⌬63 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 pRS314GU MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 YSO54 MATa trp1⌬63 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 pSO3 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 YSO55 MATa trp1⌬63 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 ⌬dbr1::HIS3 pRS314GU MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 ⌬dbr1::HIS3 YSO56 MATa trp1⌬63 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 ⌬dbr1::HIS3 pSO3 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 ⌬dbr1::HIS3 YSO57 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 pSO3 YSO2-1 YSO58 MATa trp1⌬1 his3⌬200 leu2⌬1 ura3-167 ⌬U24::kanMX4 ⌬dbr1::HIS3 pSO3 YSO3-1 S. cerevisiae RP51A intron, using plasmid pHZ18 (Teem & Rosbash, 1983) as template+ For construction of plasmid pSO4, the PCR product was digested with BsaA I and EcoR V to generate a 289-bp fragment and cloned into the Mam I site of plasmid pSO2, within the BEL1 intron, upstream of the U24 coding region+ The pSO5 plasmid was constructed with the same PCR product, digested with BsaA I and BstX I, blunt-ended with T4 DNA polymerase to generate a 156-bp fragment, and cloned into the same Mam I site in pSO2+ pKN107 is a plasmid with C. elegans dbr-1(ϩ) cDNA cloned into pRS314GU (Nam et al+, 1997), and pKN126 is a plasmid with S. pombe dbr1 ϩ cDNA cloned into pRS316GU (K+ Nam & J+D+ Boeke, unpubl+ data)+…”

Cartography of Ribosomal Proteins of the 30S Subunit from the Halophilic Haloarcula Marismortui and Complete Sequence Analysis of Protein HS26

“…The similarities concern the aminoterminal regions of the S17 proteins so it appears that this part of the 5equence must play a common functional role in the translation. For protein S17 from Saccharomyces cerevisiue, it was shown that it may play an active role in cell proliferation (Teem and Rosbash, 1983) but the function of the other proteins of this family is not yet known.…”