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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 analysis of decay of stable ΔermC derivative (left) and stable ΔermC derivative with scRNA terminator sequence (right). Times (minutes) after rifampicin addition are indicated above each lane. The replacement of the ΔermC terminator with the scRNA terminator results in an increase in size of ∼15 nt. The half-lives for the two RNAs are shown below the blot (average of three experiments).
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Figure 4: Northern blot analysis of decay of stable ΔermC derivative (left) and stable ΔermC derivative with scRNA terminator sequence (right). Times (minutes) after rifampicin addition are indicated above each lane. The replacement of the ΔermC terminator with the scRNA terminator results in an increase in size of ∼15 nt. The half-lives for the two RNAs are shown below the blot (average of three experiments).

Mentions: We tested whether the native 3′ end of scRNA was a possible initiation site for 3′ exonuclease processing. The scRNA 3′ end sequence (nts 327–354) is predicted to form a stable stem-loop structure (Figure 1) with a free energy of −14.2 kcal mol−1, which is typical of a transcription terminator sequence. We wished to determine whether the scRNA terminator structure could function as a barrier to 3′ exonuclease processivity in vivo, thus making it unlikely that 3′ exonuclease processing commences at the native 3′ end. For this experiment, the scRNA terminator structure was used to replace the 3′ terminator structure of a known stable mRNA. We have constructed a derivative of ΔermC mRNA that is quite stable, due to the presence of a strong 3′ transcription terminator structure, which has a predicted free energy of −22 kcal mol−1, and an inserted 5′-terminal secondary structure [‘ΔermC + 14/7A’ in (18)]. Because this mRNA is protected at both the 5′ and 3′ ends, it has a long half-life of more than 20 min. We reasoned that, if the scRNA 3′ end structure was a poor barrier to 3′ exonuclease processivity, replacing the 3′ terminator structure of the stable ΔermC mRNA with the 3′ terminator structure of scRNA would result in an unstable RNA, since protection at the 5′ end would be rendered irrelevant by rapid degradation from the 3′ end. Such a chimeric mRNA was constructed, and northern blot analysis was used to assess mRNA half-life (Figure 4). A small but not significant difference in the half-lives was observed, indicating that the scRNA 3′ end provides a strong barrier to 3′ exonuclease activity as does the ΔermC 3′ end. Thus, we hypothesized that 3′ exonucleolytic processing of scRNA begins at an endonuclease cleavage site, located in the 3′-proximal portion of scRNA, downstream of the Bs-RNase III b site. The approximate location of this site is labeled site X in Figure 1.Figure 4.


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 analysis of decay of stable ΔermC derivative (left) and stable ΔermC derivative with scRNA terminator sequence (right). Times (minutes) after rifampicin addition are indicated above each lane. The replacement of the ΔermC terminator with the scRNA terminator results in an increase in size of ∼15 nt. The half-lives for the two RNAs are shown below the blot (average of three experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1935012&req=5

Figure 4: Northern blot analysis of decay of stable ΔermC derivative (left) and stable ΔermC derivative with scRNA terminator sequence (right). Times (minutes) after rifampicin addition are indicated above each lane. The replacement of the ΔermC terminator with the scRNA terminator results in an increase in size of ∼15 nt. The half-lives for the two RNAs are shown below the blot (average of three experiments).
Mentions: We tested whether the native 3′ end of scRNA was a possible initiation site for 3′ exonuclease processing. The scRNA 3′ end sequence (nts 327–354) is predicted to form a stable stem-loop structure (Figure 1) with a free energy of −14.2 kcal mol−1, which is typical of a transcription terminator sequence. We wished to determine whether the scRNA terminator structure could function as a barrier to 3′ exonuclease processivity in vivo, thus making it unlikely that 3′ exonuclease processing commences at the native 3′ end. For this experiment, the scRNA terminator structure was used to replace the 3′ terminator structure of a known stable mRNA. We have constructed a derivative of ΔermC mRNA that is quite stable, due to the presence of a strong 3′ transcription terminator structure, which has a predicted free energy of −22 kcal mol−1, and an inserted 5′-terminal secondary structure [‘ΔermC + 14/7A’ in (18)]. Because this mRNA is protected at both the 5′ and 3′ ends, it has a long half-life of more than 20 min. We reasoned that, if the scRNA 3′ end structure was a poor barrier to 3′ exonuclease processivity, replacing the 3′ terminator structure of the stable ΔermC mRNA with the 3′ terminator structure of scRNA would result in an unstable RNA, since protection at the 5′ end would be rendered irrelevant by rapid degradation from the 3′ end. Such a chimeric mRNA was constructed, and northern blot analysis was used to assess mRNA half-life (Figure 4). A small but not significant difference in the half-lives was observed, indicating that the scRNA 3′ end provides a strong barrier to 3′ exonuclease activity as does the ΔermC 3′ end. Thus, we hypothesized that 3′ exonucleolytic processing of scRNA begins at an endonuclease cleavage site, located in the 3′-proximal portion of scRNA, downstream of the Bs-RNase III b site. The approximate location of this site is labeled site X in Figure 1.Figure 4.

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