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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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The rpsO mRNA of Escherichia coli is polyadenylated at multiple sites resulting from endonucleolytic processing and

J Haugel-Nielsen1, E Hajnsdorf, P Regnier

  • 1Institut de Biologie Physico-Chimique, Paris, France.

The EMBO Journal
|June 17, 1996
PubMed

Insights

Polyadenylation destabilizes the rpsO mRNA, which encodes ribosomal protein S15. However, RNAse III-generated fragments are an exception, showing stability despite polyadenylation, suggesting complex mRNA regulation pathways.

Area of Science:

  • Molecular Biology
  • RNA Metabolism
  • Gene Regulation

Background:

  • The rpsO mRNA, encoding ribosomal protein S15, is known to be destabilized by polyadenylation at the transcription terminator t1.
  • Polyadenylation is a key process in mRNA regulation, influencing stability and degradation.

Purpose of the Study:

  • To investigate the polyadenylation of various mRNA fragments derived from the rpsO transcript.
  • To determine the impact of polyadenylation on the stability of these mRNA fragments.
  • To elucidate the roles of specific RNases (RNase E, RNase III, polynucleotide phosphorylase, RNase II) in these processes.

Main Methods:

  • Analysis of mRNA fragments generated by endonucleolytic and exonucleolytic cleavage.
  • Assessment of polyadenylation in the absence of specific RNases (PNPase and RNase II).
  • Evaluation of mRNA stability following polyadenylation.

Main Results:

  • mRNA fragments with diverse 3' termini are polyadenylated, even without polynucleotide phosphorylase and RNase II.
  • Endonucleolytic cleavage by RNase E and RNase III, along with exonucleolytic degradation, generates these fragments.
  • Most polyadenylated mRNA fragments are destabilized, except for those produced by RNase III.
  • RNase E activity seems to limit the extent of poly(A) addition at the transcription terminator t1.

Conclusions:

  • Polyadenylation's effect on mRNA stability is complex and depends on the specific RNA species and processing nucleases.
  • RNase III plays a unique role in stabilizing certain polyadenylated mRNA fragments.
  • RNase E influences the polyadenylation process at the t1 terminator.

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