The Highly Conserved Bacterial RNase YbeY Is Essential in Vibrio cholerae, Playing a Critical Role in Virulence, Stress Regulation, and RNA Processing

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.

Download Full-text

Expression of human snRNA genes from beginning to end

Biochemical Society Transactions ◽

10.1042/bst0360590 ◽

2008 ◽

Vol 36 (4) ◽

pp. 590-594 ◽

Cited By ~ 72

Author(s):

Sylvain Egloff ◽

Dawn O'Reilly ◽

Shona Murphy

Keyword(s):

Rna Processing ◽

Critical Role ◽

Gene Promoters ◽

Sequence Element ◽

Protein Coding ◽

Pol Ii ◽

Protein Coding Genes ◽

Snrna Gene ◽

Gene Type ◽

New Findings

In addition to protein-coding genes, mammalian pol II (RNA polymerase II) transcribes independent genes for some non-coding RNAs, including the spliceosomal U1 and U2 snRNAs (small nuclear RNAs). snRNA genes differ from protein-coding genes in several key respects and some of the mechanisms involved in expression are gene-type-specific. For example, snRNA gene promoters contain an essential PSE (proximal sequence element) unique to these genes, the RNA-encoding regions contain no introns, elongation of transcription is P-TEFb (positive transcription elongation factor b)-independent and RNA 3′-end formation is directed by a 3′-box rather than a cleavage and polyadenylation signal. However, the CTD (C-terminal domain) of pol II closely couples transcription with RNA 5′ and 3′ processing in expression of both gene types. Recently, it was shown that snRNA promoter-specific recognition of the 3′-box RNA processing signal requires a novel phosphorylation mark on the pol II CTD. This new mark plays a critical role in the recruitment of the snRNA gene-specific RNA-processing complex, Integrator. These new findings provide the first example of a phosphorylation mark on the CTD heptapeptide that can be read in a gene-type-specific manner, reinforcing the notion of a CTD code. Here, we review the control of expression of snRNA genes from initiation to termination of transcription.

Download Full-text

A shape-shifting nuclease unravels structured RNA

10.1101/2021.11.30.470623 ◽

2021 ◽

Author(s):

Katarina Meze ◽

Armend Axhemi ◽

Dennis R Thomas ◽

Ahmet Doymaz ◽

Leemor Joshua-Tor

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Cold Shock ◽

Substrate Recognition ◽

Critical Role ◽

Rna Degradation ◽

Rna Turnover ◽

Linker Region ◽

Conformational Plasticity ◽

Gene Expression Levels

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.

Download Full-text

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin

Biochemical Journal ◽

10.1042/bcj20180387 ◽

2018 ◽

Vol 475 (19) ◽

pp. 3039-3055 ◽

Cited By ~ 5

Author(s):

Reema Kathuria ◽

Anish Kumar Mondal ◽

Rohan Sharma ◽

Samarjit Bhattacharyya ◽

Kausik Chattopadhyay

Keyword(s):

Vibrio Cholerae ◽

Formation Mechanism ◽

Pore Formation ◽

Critical Role ◽

Lipid Vesicles ◽

Membrane Lipid ◽

Human Erythrocytes ◽

Binding Ability ◽

Cholesterol Binding

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

Download Full-text

Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008261 ◽

2019 ◽

Vol 294 (37) ◽

pp. 13800-13810 ◽

Cited By ~ 1

Author(s):

Xuan Fang ◽

Jerzy Osipiuk ◽

Srinivas Chakravarthy ◽

Ming Yuan ◽

William M. Menzer ◽

...

Keyword(s):

Vibrio Cholerae ◽

Substrate Binding ◽

Critical Role ◽

Conserved Residue

Download Full-text

The NorR Regulon Is Critical for Vibrio cholerae Resistance to Nitric Oxide and Sustained Colonization of the Intestines

mBio ◽

10.1128/mbio.00013-12 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 43

Author(s):

Andrew M. Stern ◽

Amanda J. Hay ◽

Zhi Liu ◽

Fiona A. Desland ◽

Juan Zhang ◽

...

Keyword(s):

Nitric Oxide ◽

Mouse Model ◽

Vibrio Cholerae ◽

Critical Role ◽

Regulatory Protein ◽

Global Public Health ◽

Content Type ◽

Time Periods ◽

Microaerobic Conditions ◽

Over Time

ABSTRACT Vibrio cholerae, the cause of an often fatal infectious diarrhea, remains a large global public health threat. Little is known about the challenges V. cholerae encounters during colonization of the intestines, which genes are important for overcoming these challenges, and how these genes are regulated. In this study, we examined the V. cholerae response to nitric oxide (NO), an antibacterial molecule derived during infection from various sources, including host inducible NO synthase (iNOS). We demonstrate that the regulatory protein NorR regulates the expression of NO detoxification genes hmpA and nnrS, and that all three are critical for resisting low levels of NO stress under microaerobic conditions in vitro. We also show that prxA, a gene previously thought to be important for NO detoxification, plays no role in NO resistance under microaerobic conditions and is upregulated by H2O2, not NO. Furthermore, in an adult mouse model of prolonged colonization, hmpA and norR were important for the resistance of both iNOS- and non-iNOS-derived stresses. Our data demonstrate that NO detoxification systems play a critical role in the survival of V. cholerae under microaerobic conditions resembling those of an infectious setting and during colonization of the intestines over time periods similar to that of an actual V. cholerae infection. IMPORTANCE Little is known about what environmental stresses Vibrio cholerae, the etiologic agent of cholera, encounters during infection, and even less is known about how V. cholerae senses and counters these stresses. Most prior studies of V. cholerae infection relied on the 24-h infant mouse model, which does not allow the analysis of survival over time periods comparable to that of an actual V. cholerae infection. In this study, we used a sustained mouse colonization model to identify nitric oxide resistance as a function critical for the survival of V. cholerae in the intestines and further identified the genes responsible for sensing and detoxifying this stress.

Download Full-text

The critical role of RNA processing and degradation in the control of gene expression

FEMS Microbiology Reviews ◽

10.1111/j.1574-6976.2010.00242.x ◽

2010 ◽

Vol 34 (5) ◽

pp. 883-923 ◽

Cited By ~ 215

Author(s):

Cecília M. Arraiano ◽

José M. Andrade ◽

Susana Domingues ◽

Inês B. Guinote ◽

Michal Malecki ◽

...

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Critical Role ◽

Control Of Gene Expression

Download Full-text

Functional Analysis of the Essential GTP-Binding-Protein-Coding Gene cgtA of Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.02021-07 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4764-4771 ◽

Cited By ~ 16

Author(s):

Sangita Shah ◽

Bhabatosh Das ◽

Rupak K. Bhadra

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Genetic Analysis ◽

Vibrio Cholerae ◽

Critical Role ◽

Proper Function ◽

Protein Coding ◽

Gtp Binding ◽

Gene Coding ◽

Highly Sensitive

ABSTRACT The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by using genetic analysis that cgtA is essential in Vibrio cholerae even in a ΔrelA background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to the replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of CgtA plays a critical role in its proper function.

Download Full-text

The nucleolar antigen Nop52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis

Journal of Cell Science ◽

10.1242/jcs.112.12.1889 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1889-1900 ◽

Cited By ~ 8

Author(s):

T.M. Savino ◽

R. Bastos ◽

E. Jansen ◽

D. Hernandez-Verdun

Keyword(s):

Ribosomal Rna ◽

Rna Processing ◽

Mammalian Cells ◽

Critical Role ◽

Nucleolar Protein ◽

28S Rrna ◽

Human Homologue ◽

Nucleolar Proteins ◽

Protein B23 ◽

Ribosomal Rna Processing

We report the molecular characterization of a novel nucleolar protein, Nop52, and its subcellular distribution during the cell cycle and nucleologenesis. This protein was originally identified with human autoantibodies which were subsequently used to clone its corresponding cDNA. Transfection experiments in mammalian cells have confirmed that this cDNA encodes a nucleolar protein that accumulates in the nucleoli and at the periphery of the chromosomes. Nop52 is the putative human homologue of the yeast ribosomal RNA processing protein RRP1 which is involved in pre-rRNA processing from 27S to 25S and 5.8S. In nucleoli, Nop52 is excluded from the ribosomal RNA transcription sites, accumulates in the granular external domain and mainly colocalizes with nucleolar proteins involved in the late processing step such as hPop1 and protein B23. During the building process of the nucleolus at the end of mitosis, a sequential order was observed in the assembly of nucleolar proteins of early and late processing mainly via the prenucleolar body pathway. The order is the following: fibrillarin, nucleolin, Nop52 together with protein B23 in the prenucleolar bodies, and simultaneously with hPop1, and finally Ki-67. The evolutionary conservation of Nop52 and the lethal effects observed in gene disruption experiments, predict a critical role for Nop52 in the generation of 28S rRNA.

Download Full-text

TsrA Regulates Virulence and Intestinal Colonization in Vibrio cholerae

mSphere ◽

10.1128/msphere.01014-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Cory D. DuPai ◽

Ashley L. Cunningham ◽

Aaron R. Conrado ◽

Claus O. Wilke ◽

Bryan W. Davies

Keyword(s):

Vibrio Cholerae ◽

Virulence Genes ◽

Expression Patterns ◽

Critical Role ◽

Genetic Material ◽

Specific Protein ◽

Genomic Islands ◽

Differentially Expressed ◽

Intestinal Colonization ◽

Content Type

ABSTRACT Pathogenic strains of Vibrio cholerae require careful regulation of horizontally acquired virulence factors that are largely located on horizontally acquired genomic islands (HAIs). While TsrA, a Vibrionaceae-specific protein, is known to regulate the critical HAI virulence genes toxT and ctxA, its broader function throughout the genome is unknown. Here, we find that deletion of tsrA results in genomewide expression patterns that heavily correlate with those seen upon deletion of hns, a widely conserved bacterial protein that regulates V. cholerae virulence. This correlation is particularly strong for loci on HAIs, where all differentially expressed loci in the ΔtsrA mutant are also differentially expressed in the Δhns mutant. Correlation between TsrA and H-NS function extends to in vivo virulence phenotypes where deletion of tsrA compensates for the loss of ToxR activity in V. cholerae and promotes wild-type levels of mouse intestinal colonization. All in all, we find that TsrA broadly controls V. cholerae infectivity via repression of key HAI virulence genes and many other targets in the H-NS regulon. IMPORTANCE Cholera is a potentially lethal disease that is endemic in much of the developing world. Vibrio cholerae, the bacterium underlying the disease, infects humans utilizing proteins encoded on horizontally acquired genetic material. Here, we provide evidence that TsrA, a Vibrionaceae-specific protein, plays a critical role in regulating these genetic elements and is essential for V. cholerae virulence in a mouse intestinal model.

Download Full-text