Molecular mechanism of mRNA repression in
            trans
            by a ProQ‐dependent small RNA

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S. sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production.

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A search for the in trans role of GraL, an Escherichia coli small RNA*

Acta Biochimica Polonica ◽

10.18388/abp.2017_2562 ◽

2018 ◽

Vol 65 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

Maciej Dylewski ◽

Monika Ćwiklińska ◽

Katarzyna Potrykus

Keyword(s):

Small Rna ◽

Enteric Bacteria ◽

Clear Correlation ◽

Synthetic Lethal ◽

Cellular Processes ◽

In Trans ◽

Regulatory Loop

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL’s targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion’s activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.

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Early termination of the Shiga toxin transcript generates a regulatory small RNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006730117 ◽

2020 ◽

Vol 117 (40) ◽

pp. 25055-25065

Author(s):

Brandon M. Sy ◽

Ruiting Lan ◽

Jai J. Tree

Keyword(s):

Small Rna ◽

Shiga Toxin ◽

High Cell Density ◽

Shiga Toxins ◽

General Stress Response ◽

Short Rnas ◽

Short Transcript ◽

Uremic Syndrome ◽

In Trans ◽

Late Transcript

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

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