PMC

Introns accumulate in an RNase P mutant strain. Examples are shown of intron accumulation, with representations as in . (Gray lines) Introns and untranslated 5′ or 3′ segments of transcripts outside the ORFs are shown connecting ORF exons (blue boxes). (Blue arrows) Transcript starts; (black arched lines) known splicing events. Panel A does not show a blue box for the 5′ exon because the intron is part of the 5′ UTR. (A–C) Examples of introns accumulating in mRNA-coding regions with RNase P mutation, either ribosomal protein mRNA (A,B) or nonribosomal protein mRNA (C), compared to a ribosomal mRNA not containing an intron (D). An overlapping CUT in C does not show significant change in abundance at this locus.

Confirmation of pre-mRNA accumulation in an RNase P mutant strain. Total RNA was separated using 1.4% denaturing agarose gels and subjected to Northern blot analysis. Indicated mRNAs were probed with oligos specific to 3′-exon regions. Total RNA is shown for wild-type (wt) and RNase P mutant (ts) strains grown at either 30°C or 37°C. RNA size markers are indicated next to each blot (Materials and Methods). The predicted positions of pre-mRNA and mature mRNA are shown next to the bands on the blots. For Rps29A/B the pre-mRNA cartoon indicates the 5′ UTR intron. Mature mRNA levels are not significantly enriched in the RNase P mutant strain when normalized to internal Scr1 RNA control levels (values range from 0.6-fold to 1.0-fold), but new bands appear at positions predicted to be the indicated pre-mRNA. The presence of 5′ exons, as well as introns and 3′ exons, was confirmed by RT-PCR (data not shown).

Antisense RNA accumulation and overlapping mRNA de-enrichment with an RNase P mutation. Total RNA isolated from replicate cultures of wild-type (wt) or RNase P mutant (ts) samples grown at either 30°C or 37°C are shown separated on 1.4% denaturing agarose gels with subsequent Northern blot analysis. Sizes (in nucleotides, nt) were estimated from known markers (Materials and Methods), and W and C strands are as in . (A) Northern blots were probed for Sut428 or Opt2 RNAs, reprobing for Scr1 RNA as a loading control. (B) Northern blots were probed for Sut116 or Hnm1 and reprobed for Scr1 as a loading control. In both cases shown, the SUT loci accumulated RNA that differs from previous annotations, which were used for this data set (). Numbers are shown indicating different RNA species. Importantly, the RNA species most consistent with established annotation is indicated at the transcription site on the loci diagram below the Northern blots: Sut428 (1) and Sut116 (2). Also, the “sense” mRNA is shown significantly de-enriched in both panels (see also ).

Small RNAs in RNase P mutant strain. (A) Total RNA was separated on either 10% denaturing polyacrylamide gels (U1, U5, U4, U6, and 5S for loading control) or 1.4% denaturing agarose gels (U2 and Scr1) and then subjected to Northern blot analysis. RNA was probed from biological replicates of either wild-type (wt) or RNase P temperature-sensitive (ts) samples grown at either 30°C or 37°C. Fold enrichment (ts/wt) for snRNA relative to loading controls showed only modest increases in multiple experiments (1.2-fold to 2.3-fold enrichment). However, these RNAs consistently appear 2–3 nt shorter in the RNase P mutant strain in repeated experiments. This shortening is inferred to be at the 3′ end as shown by primer extension (B). (B) Primer extension analysis of U4 and U6 RNA is shown at single-nucleotide resolution with total RNA isolated from wt and ts samples grown at either 30°C or 37°C. The RNA 5′ ends of U4 and U6 appear unchanged. (C) Quantitation of U4 and U6 RNA levels relative to 5S RNA loading control. Fold enrichment (ts/wt) for U4 is 1.4 and U6 is 1.1, which indicates that even though trimming of these RNAs is occurring, they are still stably expressed. RNA was loaded twice from biological triplicate samples on the same 8% denaturing polyacrylamide gel, followed by quantitation of bands after Northern blot with error bars indicating SEM.

Noncoding RNA accumulates in an RNase P mutant strain. RNA expression data are shown along various positions of the indicated chromosomes for the Watson (W, top) and the Crick (C, bottom) strands. Abundance data across the whole genome are found in a searchable online database (see Materials and Methods). Normalized signal intensities are shown for indicated samples. Biological replicates for either wild-type (wt) or RNase P mutant (ts) strains were grown at either 30°C or 37°C in synthetic media. (Vertical red lines) Inferred positions of transcription boundaries. Genome annotations are shown in the center with annotated open reading frames (ORFs) (blue) and untranslated regions (UTRs) (gray lines). (Orange boxes) Previously annotated () SUTs; (purple boxes) CUTs; (light blue box) an uncharacterized ORF; (arrows) direction of transcription. Coordinates are indicated in base pairs in the center. (A–D) Examples of SUTs (A–C) and CUTs (B,D) accumulating in the RNase P ts strain at either 30°C or 37°C. (B,C) Reciprocal examples in which ORF RNAs decline in abundance with the RNase P mutation, while antisense RNAs accumulate. As shown in A, sometimes the inferred transcription boundaries () do not precisely align with the apparent expression in these experiments. This could reflect larger RNAs being present in the RNase P mutants.