Processing of the precursors to small nucleolar RNAs and rRNAs requires common components - PubMed (original) (raw)
Processing of the precursors to small nucleolar RNAs and rRNAs requires common components
E Petfalski et al. Mol Cell Biol. 1998 Mar.
Abstract
The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae. Here we report that these two snoRNAs are synthesized by processing of a larger common transcript. In strains mutant for two 5'-->3' exonucleases, Xrn1p and Rat1p, families of 5'-extended forms of snR190 and U14 accumulate; these have 5' extensions of up to 42 and 55 nucleotides, respectively. We conclude that the 5' ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites. In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences. Analysis of RNA extracted from a dbr1-delta strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat. In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity. The 5' ends of these snoRNAs are also generated by Xrn1p and Rat1p. The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5' end of the 5.8S rRNA. Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.
Figures
FIG. 1
Accumulation of excised pre-rRNA fragments in exonuclease mutants. (A) Northern hybridization of RNAs extracted from strains of the indicated genotypes following growth at 25°C or 2 h after transfer to 37°C. Panels: a, Riboprobe specific for the A0-A1 region; b, probe specific for the D-A2 region (oligonucleotide 002); c, riboprobe specific for the A2-A3 region; d, 5′-extended 5.8S detected with the probe for the A3-B1 region (oligonucleotide 003); e, mature 5.8S rRNA (oligonucleotide 017). RNA was separated on a gel containing 8% polyacrylamide. (B) Structure of the pre-rRNA, showing the locations of processing sites above the pre-rRNA. Lines below the pre-rRNA indicate the species detected in panels a to d of panel A. The effects of the mutations on the accumulation of the fragments referred to as a, b, c, and d are shown in panels a, b, c, and d, respectively, of panel A.
FIG. 2
Northern analysis of the synthesis of U14 and snR190. (A) Northern hybridization of RNAs extracted from strains of the indicated genotypes following growth at 25°C or 2 h after transfer to 37°C. Panels: a, 5′-extended forms of snR190 (oligonucleotide αsnR190+1); b, mature snR190 (oligonucleotide αsnR190); c, 5′-extended forms of U14 (oligonucleotide αU14-9); d, mature U14 (oligonucleotide αU14); e, mature 5S rRNA (oligonucleotide 041). RNA was separated on a gel containing 8% polyacrylamide. (B) Predicted structure of the precursor to snR190 and U14. Lines below the pre-snoRNA indicate the species detected in panels a to d of panel A.
FIG. 3
Primer extension analysis of the synthesis of U14 and snR190. 5′-extended forms of snR190 and U14 are detected in 5′→3′ exonuclease mutants. Primer extension was performed with the αU14 or αsnR190 oligonucleotide on RNA extracted from strains of the indicated genotypes following growth at 25°C or 2 h after transfer to 37°C. The lower panels show shorter exposures of the same primer extensions as the corresponding upper panels.
FIG. 4
Lariat forms of U18 and U24 accumulate in an intron-debranching mutant. (A) In RNA from the _dbr1_-Δ strain, mature U18 and U24 are underaccumulated and longer forms are detected. (B and C) RNAs extracted from the _dbr1_-Δ strain and an otherwise isogenic DBR1 strain were separated by long migration on gels containing either 6% (B) or 8% (C) polyacrylamide. Lanes + and −, DBR1 and _dbr1_-Δ strains, respectively. The slow-migrating forms of U18 and U24 differ in their relative migrations on the gels compared to the linear RNA species U3 (311 nt), U1 (568 nt), and 18S rRNA (1,860 nt). Note that mature U18 and U24 are substantially smaller than the linear RNA species shown and have been lost from the gels in panels B and C.
FIG. 5
Northern analysis of the synthesis of U18 and U24. Longer forms of U18 and U24 are detected in 5′→3′ exonuclease mutants. Northern hybridization of RNAs extracted from strains of the indicated genotypes is shown. For U18, RNA was separated on a gel containing 6% polyacrylamide; for U24, which is smaller, a gel containing 8% polyacrylamide is shown. The positions of migration of U14 (126 nt), snR190 (190 nt), snR10 (245 nt) and U3 (311 nt) determined by subsequent Northern hybridization of the same filters are indicated, as are the positions of migration of mature 5S and 5.8S rRNAs. Species predicted to be 5′- and 3′-extended forms of U24 and U18 are indicated (+5′, +3′, and +5′ +3′).
FIG. 6
Primer extension analysis of the synthesis of U24. (Left) Primer extension with a probe to mature U24 (αU24). (Right) Primer extension with an oligonucleotide (oligo) spanning the 3′ end of U24 (U24-3′fl). The positions of the mature 5′ end of U24 and the intron 5′ splice site (5′ IVS) are indicated on the DNA sequence ladder.
FIG. 7
(A) Model for the processing of pre-snR190 and pre-U14. The coding sequences of snR190 and U14 lie in the same orientation in the genome and are separated by only 67 nt (46). We propose that they are synthesized from a common precursor which extends from 302 nt 5′ to snR190 to beyond the 3′ end of U14. Cleavage of the pre-snoRNA at snR190 position −42 and U14 position −55 is envisaged to be followed by exonuclease digestion by Rat1p to the 5′ ends of the snoRNAs and 3′ trimming. (B) Model for the processing of pre-U24. U24 is encoded in the intron of the BEL1 gene (23, 35) and is generated from the excised intron lariat. Following intron debranching, processing is envisaged to consist of exonuclease digestion by Rat1p to the 5′ end of the snoRNA and 3′ trimming. IBP, intron branch point.
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