Related Experiment Video

Updated: Nov 25, 2025

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
06:49

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Published on: May 8, 2020

4.9K

Polynucleotide phosphorylase and RNA helicase CshA cooperate in Bacillus subtilis mRNA decay

Shakti Ingle1, Shivani Chhabra1, Denise Laspina1

  • 1Icahn School of Medicine at Mount Sinai, Department of Pharmacological Sciences, New York, NY, USA.

RNA Biology
|December 16, 2020
PubMed

Insights

Polynucleotide phosphorylase (PNPase) degrades mRNA in Bacillus subtilis. Its activity is hindered by RNA structures, requiring the RNA helicase CshA for efficient mRNA turnover, especially for structured RNAs.

Area of Science:

  • Molecular Biology
  • Microbiology
  • RNA Metabolism

Background:

  • Polynucleotide phosphorylase (PNPase) is the primary 3' exoribonuclease responsible for mRNA degradation in Bacillus subtilis.
  • PNPase activity is inhibited by strong RNA secondary structures in vitro.
  • Efficient in vivo mRNA turnover is hypothesized to involve RNA helicases working with PNPase.

Purpose of the Study:

  • To investigate the role of the RNA helicase CshA in Bacillus subtilis mRNA decay mediated by PNPase.
  • To determine how RNA secondary structures influence PNPase processivity in vivo.
  • To identify potential RNA helicases involved in vegetative growth mRNA decay.

Main Methods:

  • Development of a novel colorimetric screen for monitoring mRNA decay in Bacillus subtilis using slrA mRNA.
  • Analysis of PNPase processivity on various small, monocistronic mRNAs with differing predicted RNA structures.
  • Northern blot analysis to confirm PNPase activity and the impact of CshA and RNA structure.

Main Results:

  • PNPase processivity varied depending on RNA sequence and the presence of CshA.
  • A strong correlation was observed between predicted stem-loop structure strength and impaired PNPase processivity.
  • Internal RNA structures significantly hindered PNPase activity, with a more pronounced effect in the absence of CshA.

Conclusions:

  • Efficient 3' exonucleolytic decay of Bacillus subtilis RNA is dependent on the combined action of PNPase and CshA.
  • CshA is crucial for overcoming inhibitory RNA secondary structures during mRNA degradation.
  • Other identified Bacillus subtilis RNA helicases are not significantly involved in mRNA decay during vegetative growth.
Keywords:
3ʹ exoribonucleaseBacillus subtilisCshAPNPaseRNA helicasemRNA decay

More Related Videos

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events10:59

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Published on: May 13, 2019

10.0K
Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC09:15

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

Published on: May 9, 2020

5.4K

Related Concept Videos

Coordination of Gene Expression Processes in Bacteria01:29

Coordination of Gene Expression Processes in Bacteria

The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...
397
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
31.7K
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

11.1K
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
11.3K
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

3.1K
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
6.1K