scholarly journals Initiation of Decay of Bacillus subtilis rpsO mRNA by Endoribonuclease RNase Y

2010 ◽  
Vol 192 (13) ◽  
pp. 3279-3286 ◽  
Author(s):  
Shiyi Yao ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Decay ◽  
Inducible Promoter ◽  
Transcription Unit ◽  
The Body ◽  
Rate Determining Step ◽  
Ribosomal Protein S15 ◽  
Mrna Half Life ◽  
Rnase Y ◽  
Specific Mrna

ABSTRACT rpsO mRNA, a small monocistronic mRNA that encodes ribosomal protein S15, was used to study aspects of mRNA decay initiation in Bacillus subtilis. Decay of rpsO mRNA in a panel of 3′-to-5′ exoribonuclease mutants was analyzed using a 5′-proximal oligonucleotide probe and a series of oligonucleotide probes that were complementary to overlapping sequences starting at the 3′ end. The results provided strong evidence that endonuclease cleavage in the body of the message, rather than degradation from the native 3′ end, is the rate-determining step for mRNA decay. Subsequent to endonuclease cleavage, the upstream products were degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5′ exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.

scholarly journals Effect of Translational Signals on mRNA Decay in Bacillus subtilis

2003 ◽  
Vol 185 (18) ◽  
pp. 5372-5379 ◽  
Author(s):  
Josh S. Sharp ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Stability ◽  
Half Life ◽  
Ternary Complex ◽  
Mrna Decay ◽  
Peptide Bond ◽  
Start Codon ◽  
Transcription Terminator ◽  
Ribosome Binding ◽  
Mrna Half Life

ABSTRACT A 254-nucleotide model mRNA, designated ΔermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. ΔermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure. Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. Data for the decay of ΔermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation—even the formation of the first peptide bond—was not important for stability. The half-life of an untranslated 3.2-kb ΔermC-lacZ fusion RNA was similar to that of a translated ΔermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5′-end-dependent activity, possibly a 5′-binding endonuclease, which is required for the initiation of mRNA decay. This model is supported by the finding that increasing the distance from the 5′ end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life. These results underscore the importance of the 5′ end to mRNA stability in B. subtilis.

scholarly journals Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation

2021 ◽  
Vol 9 (5) ◽  
pp. 1046
Author(s):  
Inam Ul Haq ◽  
Sabine Brantl
Keyword(s):  
Bacillus Subtilis ◽  
Antisense Rna ◽  
Rna Binding ◽  
Rna Degradation ◽  
Metabolic Enzyme ◽  
Dual Function ◽  
Small Proteins ◽  
Moonlighting Proteins ◽  
Rnase Y

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

Probing Drosophila gene function by antisense RNA

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 422-425 ◽  
Author(s):  
Reinhard Schuh ◽  
Herbert Jäckle
Keyword(s):  
Antisense Rna ◽  
Germ Line ◽  
Conventional Technique ◽  
Transcription Unit ◽  
The Body ◽  
Mutant Phenotype ◽  
Drosophila Embryo ◽  
Alternative Approach ◽  
Pattern Forming ◽  
Germ Line Transformation

The conventional technique for assigning a particular genetic function to a cloned transcription unit has relied on the rescue of the mutant phenotype by germ line transformation. An alternative approach is to mimic a mutant phenotype by the use of antisense RNA injections to produce phenocopies. This approach has been successfully used to identify genes involved in early pattern forming processes in the Drosophila embryo. At the time when antisense RNA is injected, the embryo develops as a syncytium composed of about 5000 nuclei which share a common cytoplasm. The gene interactions required to establish the body plan occur before cellularization at the blastoderm stage. Thus the nuclei and their exported transcripts are accessible to the injected antisense RNA. The antisense RNA interferes with the endogenous RNA by an as yet unidentified mechanism. The extent of interference is only partial and produces phenocopies with characteristics of weak mutant alleles. In our lab and others, this approach has been successfully used to identify several genes required for normal Drosophila pattern formation.Key words: Drosophila segmentation, phenocopy, antisense RNA, Krüppel gene.

Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor

1993 ◽  
Vol 13 (4) ◽  
pp. 2411-2419
Author(s):  
K P Mann ◽  
E A Weiss ◽  
J R Nevins
Keyword(s):  
Early Phase ◽  
Late Phase ◽  
Transcription Unit ◽  
Adenovirus Infection ◽  
Processing Factor ◽  
Proximal Half ◽  
Rate Determining Step ◽  
Frequency Of Use ◽  
Late Transcription ◽  
A Site

The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection.

Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation

1984 ◽  
Vol 4 (5) ◽  
pp. 875-882
Author(s):  
M J Imperiale ◽  
J R Nevins
Keyword(s):  
Transcription Initiation ◽  
Essential Element ◽  
Inducible Promoter ◽  
Initiation Site ◽  
Transcription Unit ◽  
Upstream Sequence ◽  
Wild Type ◽  
Promoter Sequences ◽  
Adenovirus 5 ◽  
Wild Type Promoter

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

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

RNA Biology ◽  
2020 ◽  
pp. 1-10
Author(s):  
Shakti Ingle ◽  
Shivani Chhabra ◽  
Denise Laspina ◽  
Elizabeth Salvo ◽  
Bo Liu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Decay ◽  
Rna Helicase ◽  
Polynucleotide Phosphorylase

Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery

Vaccine ◽  
2006 ◽  
Vol 24 (15) ◽  
pp. 2935-2943 ◽  
Author(s):  
Juliano D. Paccez ◽  
Wilson B. Luiz ◽  
Maria E. Sbrogio-Almeida ◽  
Rita C.C. Ferreira ◽  
Wolfgang Schumann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Expression System ◽  
Inducible Promoter ◽  
Antigen Delivery

scholarly journals Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hamouche ◽  
Cyrille Billaudeau ◽  
Anna Rocca ◽  
Arnaud Chastanet ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Model Organism ◽  
Living Cells ◽  
Cell Periphery ◽  
Rnase E ◽  
Content Type ◽  
Endonucleolytic Cleavage ◽  
Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

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