| 1. |
- Abrhámová, Kateřina, et al.
(author)
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Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.
- 2024
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In: RNA Biology. - 1547-6286 .- 1555-8584. ; 21:1, s. 1-17
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Journal article (peer-reviewed)abstract
- Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.
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| 2. |
- Alm Rosenblad, Magnus, 1957, et al.
(author)
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The enigmatic RNase MRP of kinetoplastids
- 2021
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In: Rna Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 18:Supplement 1, s. 139-147
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Journal article (peer-reviewed)abstract
- The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.
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| 3. |
- Avesson, Lotta, et al.
(author)
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Abundant class of non-coding RNA regulates development in the social amoeba Dictyostelium discoideum
- 2011
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In: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 8:6, s. 1094-1104
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Journal article (peer-reviewed)abstract
- Non-coding (nc)RNAs are important players in most biological processes. Although small RNAs such as microRNAs and small interfering RNAs have emerged as exceptionally important regulators of gene expression, great numbers of larger ncRNAs have also been identified. Many of these are abundant and differentially expressed but their functions have in most cases not been elucidated. The social amoeba Dictyostelium discoideum contain the ncRNAs commonly found in eukaryotes. In addition, we previously reported the identification of two novel classes of 42-65 nt long stem-loop forming RNAs, Class I and Class II RNAs, with unknown function. In this study we have further characterized these abundant ncRNAs, which are down regulated during development. We have confirmed expression of 29 Class I RNAs and experimentally verified the formation of the computationally predicted short conserved stem structure. Furthermore, we have for the first time created knockout strains for several small ncRNA genes in D. discoideum and found that deletion of one of the Class I RNAs, DdR-21, results in aberrant development. In addition we have shown that this Class I RNA forms a complex with one or several proteins but do not appear to be associated with ribosomes or polysomes. In a pull down assay, several proteins interacting with DdR-21 were identified, one of these has two RNA recognition motifs (RRMs). The purified RRM containing protein was demonstrated to bind directly and specifically to DdR-21.
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| 4. |
- Bao, Letian, et al.
(author)
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Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre
- 2024
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In: RNA Biology. - : Taylor & Francis. - 1547-6286 .- 1555-8584. ; 21:1, s. 31-41
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Journal article (peer-reviewed)abstract
- Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.
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| 5. |
- Bentolila, Stéphane, et al.
(author)
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Quantitative trait locus mapping identifies REME2, a PPR-DYW protein required for editing of specific C targets in Arabidopsis mitochondria.
- 2013
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In: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 10:9, s. 1520-5
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Journal article (peer-reviewed)abstract
- Targeted RNA editing by C-to-U alteration occurs at hundreds of sites in the mitochondrial transcriptome of flowering plants. By using natural variation and positional cloning on a population of Arabidopsis recombinant inbred lines between the ecotypes Col and Ler, we found that two of these occurrences involve the Arabidopsis PPR-DYW protein REME2 (Required for Efficiency of Mitochondrial Editing2). The analysis of a knockdown mutant along with silenced tissues confirms the specificity of REME2 for both sites located in mitochondrial ribosomal protein genes (rps3-1534 and rps4-175). The conservation level of both cis elements is relatively high, as is the amino acid conservation among flowering plants for both genes in that location, underlining the importance of these editing events and REME2.
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| 6. |
- Broglia, Laura, et al.
(author)
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RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B
- 2018
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In: RNA Biology. - : Taylor & Francis. - 1547-6286 .- 1555-8584. ; 15:10, s. 1336-1347
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Journal article (peer-reviewed)abstract
- Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.
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| 7. |
- Bäreclev, Caroline, et al.
(author)
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DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression
- 2014
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In: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 11:11, s. 1458-1467
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Journal article (peer-reviewed)abstract
- RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37 degrees C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26 degrees C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20 degrees C, whereas the effect was marginal at 37 degrees C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.
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| 8. |
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| 9. |
- Chylinski, Krzysztof, et al.
(author)
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The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
- 2013
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In: RNA Biology. - : Landes Bioscience. - 1547-6286 .- 1555-8584. ; 10:5, s. 726-737
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Journal article (peer-reviewed)abstract
- CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.
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| 10. |
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