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Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site.

Yao S, Blaustein JB, Bechhofer DH - Nucleic Acids Res. (2007)

Bottom Line: Bs-RNase III was found to cleave precursor scRNA at two sites (the 5' and 3' cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3' exoribonuclease to the mature scRNA.RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences.The subsequent exonucleolytic processing is carried out largely by RNase PH.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai School of Medicine of New York University, New York, NY 10029, USA.

ABSTRACT
Small cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNase III-like endoribonuclease, Bs-RNase III, in scRNA processing. Bs-RNase III was found to cleave precursor scRNA at two sites (the 5' and 3' cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3' exoribonuclease to the mature scRNA. Here we show that Bs-RNase III cleaves primarily at the 5' cleavage site and inefficiently at the 3' site. RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNase PH.

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Northern blot analyses of scRNA processing products using different oligonucleotide probes. In panels A–C, RNA from the following strains was analyzed: wt, wild type; quad, quadruple exonuclease mutant; rncS, rncS deletion strain. The probe used is written below each blot, together with a schematic diagram of scRNA on the left side of the blot showing with bold lines the extent of complementarity to each probe. Sequencing reaction that provided the nucleotide size marker is at left. Arrowheads with sizes of bands detected are on the right, together with schematic diagrams of the RNAs detected. In panel D, the 3′-terminal probe was used to detect a downstream fragment of endonuclease cleavage in the wild-type strain.
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Figure 6: Northern blot analyses of scRNA processing products using different oligonucleotide probes. In panels A–C, RNA from the following strains was analyzed: wt, wild type; quad, quadruple exonuclease mutant; rncS, rncS deletion strain. The probe used is written below each blot, together with a schematic diagram of scRNA on the left side of the blot showing with bold lines the extent of complementarity to each probe. Sequencing reaction that provided the nucleotide size marker is at left. Arrowheads with sizes of bands detected are on the right, together with schematic diagrams of the RNAs detected. In panel D, the 3′-terminal probe was used to detect a downstream fragment of endonuclease cleavage in the wild-type strain.

Mentions: (A) Schematic diagram of scRNA with nucleotide sequence of 5′ and 3′ ends. The large internal loop of 255 nt is not drawn to scale. Solid arrows show location of Bs-RNase III cleavage sites A and b. Proposed cleavage site X is indicated by dashed arrow. Asterisk denotes 3′ end of mature scRNA. The bold C and A nucleotides are at the 3′ end of the 279- and 283-nt products, respectively. Boxed nucleotides mark the ends of the sequence that is complementary to the 3′-proximal oligonucleotide probe (shown schematically in Figure 6B). Circled nucleotides mark the ends of the sequence that is complementary to the 3′-terminal oligonucleotide probe (shown schematically in Figure 6C and D). (B) Line diagrams of RNA products detected by various probes. Arrowheads above full-length line indicate sites of Bs-RNase III cleavage. Diamond below full-length line indicates site X. Numbers at each end of the line indicate 5′ and 3′ ends of RNAs. Above each line is the likely origin of the RNA species, with the size of the RNA in parentheses. (The sizes of the RNAs are those predicted from earlier data and calculated from the data shown in Figures 2, 3 and 6. The degree of precision from these data is ±2 nt, as mentioned in the text.) For clarity, the 314- and 310-nt RNAs, as well as the 283- and 279-nt RNAs, are grouped.


Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site.

Yao S, Blaustein JB, Bechhofer DH - Nucleic Acids Res. (2007)

Northern blot analyses of scRNA processing products using different oligonucleotide probes. In panels A–C, RNA from the following strains was analyzed: wt, wild type; quad, quadruple exonuclease mutant; rncS, rncS deletion strain. The probe used is written below each blot, together with a schematic diagram of scRNA on the left side of the blot showing with bold lines the extent of complementarity to each probe. Sequencing reaction that provided the nucleotide size marker is at left. Arrowheads with sizes of bands detected are on the right, together with schematic diagrams of the RNAs detected. In panel D, the 3′-terminal probe was used to detect a downstream fragment of endonuclease cleavage in the wild-type strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1935012&req=5

Figure 6: Northern blot analyses of scRNA processing products using different oligonucleotide probes. In panels A–C, RNA from the following strains was analyzed: wt, wild type; quad, quadruple exonuclease mutant; rncS, rncS deletion strain. The probe used is written below each blot, together with a schematic diagram of scRNA on the left side of the blot showing with bold lines the extent of complementarity to each probe. Sequencing reaction that provided the nucleotide size marker is at left. Arrowheads with sizes of bands detected are on the right, together with schematic diagrams of the RNAs detected. In panel D, the 3′-terminal probe was used to detect a downstream fragment of endonuclease cleavage in the wild-type strain.
Mentions: (A) Schematic diagram of scRNA with nucleotide sequence of 5′ and 3′ ends. The large internal loop of 255 nt is not drawn to scale. Solid arrows show location of Bs-RNase III cleavage sites A and b. Proposed cleavage site X is indicated by dashed arrow. Asterisk denotes 3′ end of mature scRNA. The bold C and A nucleotides are at the 3′ end of the 279- and 283-nt products, respectively. Boxed nucleotides mark the ends of the sequence that is complementary to the 3′-proximal oligonucleotide probe (shown schematically in Figure 6B). Circled nucleotides mark the ends of the sequence that is complementary to the 3′-terminal oligonucleotide probe (shown schematically in Figure 6C and D). (B) Line diagrams of RNA products detected by various probes. Arrowheads above full-length line indicate sites of Bs-RNase III cleavage. Diamond below full-length line indicates site X. Numbers at each end of the line indicate 5′ and 3′ ends of RNAs. Above each line is the likely origin of the RNA species, with the size of the RNA in parentheses. (The sizes of the RNAs are those predicted from earlier data and calculated from the data shown in Figures 2, 3 and 6. The degree of precision from these data is ±2 nt, as mentioned in the text.) For clarity, the 314- and 310-nt RNAs, as well as the 283- and 279-nt RNAs, are grouped.

Bottom Line: Bs-RNase III was found to cleave precursor scRNA at two sites (the 5' and 3' cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3' exoribonuclease to the mature scRNA.RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences.The subsequent exonucleolytic processing is carried out largely by RNase PH.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai School of Medicine of New York University, New York, NY 10029, USA.

ABSTRACT
Small cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNase III-like endoribonuclease, Bs-RNase III, in scRNA processing. Bs-RNase III was found to cleave precursor scRNA at two sites (the 5' and 3' cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3' exoribonuclease to the mature scRNA. Here we show that Bs-RNase III cleaves primarily at the 5' cleavage site and inefficiently at the 3' site. RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNase PH.

Show MeSH
Related in: MedlinePlus