Designing RNA structures

Abstract Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5′UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.

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Conserved long-range base pairings are associated with pre-mRNA processing of human genes

Nature Communications ◽

10.1038/s41467-021-22549-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Svetlana Kalmykova ◽

Marina Kalinina ◽

Stepan Denisov ◽

Alexey Mironov ◽

Dmitry Skvortsov ◽

...

Keyword(s):

Long Range ◽

Rna Folding ◽

Current Knowledge ◽

Rna Structures ◽

Base Pairs ◽

Protein Coding ◽

Proximity Ligation ◽

Transcriptional Suppression ◽

Human Genes ◽

Cleavage And Polyadenylation

AbstractThe ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3’-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

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Mapping circular RNA structures in living cells by SHAPE-MaP

Methods ◽

10.1016/j.ymeth.2021.01.011 ◽

2021 ◽

Author(s):

Si-Kun Guo ◽

Fang Nan ◽

Chu-Xiao Liu ◽

Li Yang ◽

Ling-Ling Chen

Keyword(s):

Circular Rna ◽

Living Cells ◽

Rna Structures

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Activation of MDA5 Requires Higher-Order RNA Structures Generated during Virus Infection

Journal of Virology ◽

10.1128/jvi.00770-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10761-10769 ◽

Cited By ~ 275

Author(s):

Andreas Pichlmair ◽

Oliver Schulz ◽

Choon-Ping Tan ◽

Jan Rehwinkel ◽

Hiroki Kato ◽

...

Keyword(s):

Higher Order ◽

Encephalomyocarditis Virus ◽

Signaling Cascade ◽

Rna Structures ◽

Innate Response ◽

Double Stranded Rna ◽

Infected Cells ◽

Alpha Beta ◽

Complementary Rnas ◽

Inducible Gene

ABSTRACT Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-α/β) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-α/β production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-α/β production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.

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Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6337-6349 ◽

Cited By ~ 27

Author(s):

S E Wells ◽

M Ares

Keyword(s):

Synthetic Lethality ◽

Small Nuclear Rna ◽

Rna Structures ◽

Stem Loop ◽

U2 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

Nuclear Rna ◽

Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

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