RNAse III-Mediated Degradation of Unspliced Pre-mRNAs and Lariat Introns  Michal Danin-Kreiselman, Chrissie Young Lee, Guillaume Chanfreau  Molecular Cell  Volume 11, Issue 5, Pages 1279-1289 (May 2003) DOI: 10.1016/S1097-2765(03)00137-0

Figure 1 Yeast RNase III Cleaves Unspliced RPS22B Precursors and Lariat Intron 1 (A) Structure of the RPS22B gene. Exon sequences are represented by rectangles while intronic sequences are represented by lines. The smaller rectangle represents the snR44 snoRNA. (B) Analysis of the expression of the RPS22B gene in wild-type, rnt1Δ, dbr1Δ, rnt1Δ dbr1Δ, and prp2-ts strains. Membranes were hybridized to probes hybridizing to intron 1, intron 2, or exon 3 regions of RPS22B, stripped, and hybridized with the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control. The different products are indicated on the side of the membrane. The apparent increase in the level of the snR44 snoRNA in the rnt1Δ samples is due to lower amounts of rRNA in these samples. Accordingly, other abundant RNAs such as snoRNAs appear more abundant in the rnt1Δ samples when equal masses of RNA (10 μg) are loaded. Molecular Cell 2003 11, 1279-1289DOI: (10.1016/S1097-2765(03)00137-0)

Figure 2 Detection of Rnt1p Cleavage Products in Exonuclease Mutant Strains (A) Detection of Rnt1p cleavage products in a xrn1Δ rat1-1 exonuclease mutant strain. Northern blot analysis was performed on RNAs extracted from the indicated strains grown either at 25°C or shifted at 37°C for 2 hr 30 min. The probes used are the same as in Figure 1B. The cleaved products are indicated by the small arrows perpendicular to the product symbol. (B) Analysis of exonuclease single-mutant strains. Legends are as in Figure 2A. A sample extracted from a xrn1Δ rat1-1 mutant strain shifted at 37°C was included for comparison. (C) Detection of the upstream Rnt1p cleavage product in vivo using the rrp6Δ exosome mutant strain. Northern blot analysis was performed on RNAs extracted from the indicated strains grown at 25°C. The UP probe hybridizes to nucleotides 12–346 of the intron. The DS probe hybridizes to nucleotides 245–537 of the intron. Molecular Cell 2003 11, 1279-1289DOI: (10.1016/S1097-2765(03)00137-0)

Figure 3 Rnt1p Cleaves a dsRNA Region within the First Intron of RPS22B (A) Mapping of the cleavage site in vivo using exonucleases mutant strains. Primer extension was performed on RNAs extracted from the indicated strains as in Figure 1C. Each primer extension included a 5′ end-labeled primer specific for the first intron of RPS22B and a 5′ end labeled primer hybridizing to the snoRNA snR37 as a positive control. The sequencing lane was generated with the intron 1 primer. (B) Secondary structure folding of the intronic region near the cleavage sites. The structure shown is the most stable structure calculated using Mfold. The empty triangle indicates the location of the cleavage site detected in vivo at 37°C in Figure 3A, while the black triangles indicate the locations of the cleavage sites detected in vitro in Figure 3C. The gray triangle indicates the location of the cleavage site detected in vivo at 25°C, which may correspond to a partially trimmed species due to partial inactivation of Rat1p at this temperature. The size of each triangle is approximately proportional to the intensity of the bands observed. (C) Mapping of the cleavage site in vitro using a model substrate and recombinant Rnt1p. In vitro transcribed RNAs were incubated with buffer alone (lane1), recombinant wild-type (lane 3), or mutant (E320K, lane 2) Rnt1p and used as primer extension templates. (D) Northern blot analysis of strains carrying deletions of the stem-loop sequences. Analysis of RNAs extracted from WT and rnt1Δ strains and from strains carrying the deletion of stem-loop 1 (SL1Δ) or of both stem-loops (SL1+2Δ), and these stem-loop deletions combined with the DBR1 deletion. Membranes were hybridized either to the RPS22B exon 3 probe or to the RPS22B intron 1 probe, and then to the G3PDH control. Molecular Cell 2003 11, 1279-1289DOI: (10.1016/S1097-2765(03)00137-0)

Figure 4 Northern Blot Analysis of Intron-Containing Yeast Transcripts in Wild-Type, rnt1Δ, dbr1Δ, and rnt1Δ dbr1Δ Strains Legends are as in Figure 1B. All probes span the intron and the exon 2 regions, unless specified in the figure. Molecular Cell 2003 11, 1279-1289DOI: (10.1016/S1097-2765(03)00137-0)

Figure 5 Rnt1p Cleaves a dsRNA Region within the RPL18A Intron (A) Detection of the downstream Rnt1p cleavage product in vivo using 5′→3′ exonuclease mutant strains. Legends as in Figures 2A and 2B, except that RPL18A probes were used. (B) Detection of the upstream Rnt1p cleavage product in vivo using the rrp6Δ mutant strain. Legends as in Figure 2C, except that RPL18A probes were used. The UP probe hybridizes from 57 nucleotides upstream from the AUG to nucleotide 201 of the intron. The DS probe hybridizes from nucleotide 160 of the intron to 8 nucleotides downstream from the stop codon. (C) Mapping of the cleavage site in vivo by primer extension using exonuclease mutant strains. Legends as in Figure 3A, except that an RPL18A intronic primer was used. (D) Secondary structure folding of the RPL18A intronic region near the cleavage site. The arrow indicates the cleavage site observed in vivo in Figure 5B. Molecular Cell 2003 11, 1279-1289DOI: (10.1016/S1097-2765(03)00137-0)