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Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

Acta Crystallogr F Struct Biol Commun. 2015 Jun 1;71(Pt 6):735-740

Authors: Osawa T, Inanaga H, Numata T

Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

PMID: 26057804 [PubMed – as supplied by publisher]

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Small RNAs, 5′ UTR elements and RNA-binding proteins in intracellular bacteria: impact on metabolism and virulence.

Small RNAs, 5′ UTR elements and RNA-binding proteins in intracellular bacteria: impact on metabolism and virulence.

FEMS Microbiol Rev. 2015 May;39(3):331-349

Authors: Oliva G, Sahr T, Buchrieser C

Abstract
Sequencing-based studies have illuminated increased transcriptional complexity within the genome structure of bacteria and have resulted in the identification of many small regulatory RNAs (sRNA) and a large amount of antisense transcription. It remains an open question whether these sRNAs all indeed play regulatory roles, but their identification led to an exponential increase in studies searching for their function. This allowed to show that sRNAs may modulate virulence gene expression, cellular differentiation, metabolic functions, adaptation to environmental conditions and pathogenesis. In this review we will provide mechanistic insights into how sRNAs bind mRNAs and/or proteins. Furthermore, the important roles of the RNA chaperone Hfq, the CsrA system and the CRISPR RNA will be discussed. We will then focus on sRNAs and 5(‘) untranslated region (UTR) elements of intracellular bacteria like Chlamydia, Listeria, Legionella, or Salmonella, and place emphasis on those that are expressed during replication in host cells and are implicated in virulence and metabolism. In addition, sRNAs that regulate motility, iron homeostasis, and differentiation or stress responses will be highlighted. Taken together sRNAs constitute key elements in many major regulatory networks governing the intracellular life and virulence of pathogenic bacteria.

PMID: 26009640 [PubMed – as supplied by publisher]

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3D Animation of MicroRNA biogenesis and functions | miRNA Blog

3D Animation of MicroRNA biogenesis and functions | miRNA Blog.

SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma.

SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma.

Sci Rep. 2015;5:8588

Authors: Chen L, Han L, Wei J, Zhang K, Shi Z, Duan R, Li S, Zhou X, Pu P, Zhang J, Kang C

Abstract
Glioblastoma (GBM) is associated with disproportionately high morbidity and mortality, reflecting the need to develop new diagnostic and therapeutic targets for this disease. Recently, accumulating evidence has suggested that small nucleolar RNAs (snoRNAs) are gaining prominence and are more actively involved in tumorigenesis than previously thought. However, no report concerning the implication of snoRNAs in glioma has been published to date. In our study, SNORD76 was first found to be inversely associated with Hox Transcript Antisense Intergenic RNA (HOTAIR) knockdown, and surprisingly, forcibly expressed SNORD76 inhibited proliferation and growth of glioma cells. Moreover, downregulation of SNORD76 led to a more malignant phenotype. The pleiotropy of SNORD76 overexpression could be achieved at least partially through inducing cell cycle arrest at S phase by affecting the Rb-associated cell cycle regulation. Enforced SNORD76 expression in orthotopic tumors resulted in decreased tumor growth and the reduction of tumor volume. Additionally, in surgically resected glioma tissues, SNORD76, not its host gene, was associated with the WHO classification and was selectively downregulated in GBM (WHO grade IV). Collectively, our study adds to a growing body of evidence for the participation of snoRNAs in gliomagenesis and is the first to implicate a snoRNA in glioblastoma.

PMID: 25715874 [PubMed – in process]

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CSHLIn a role reversal, RNAs proofread themselves | News & Features

CSHLIn a role reversal, RNAs proofread themselves | News & Features

CSHLIn a role reversal, RNAs proofread themselves | News & Features.

Forget the Selfish Gene: Evolution of Life Is Driven by the Selfish Ribosome, Research Suggests

Since the discovery of how DNA encodes genetic information, most research on the evolution of life has focused on genes. According to the ‘selfish gene’ theory, cells and organisms exist … Jan. 7, 2015 

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Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.

Related Articles

Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.

Nature. 2014 Nov 6;515(7525):147-50

Authors: Zhao H, Sheng G, Wang J, Wang M, Bunkoczi G, Gong W, Wei Z, Wang Y

Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5′ and 3′ terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long β-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.

PMID: 25118175 [PubMed – indexed for MEDLINE]

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tRFs and the Argonautes: gene silencing from antiquity – BioMed Central blog

tRFs and the Argonautes: gene silencing from antiquity – BioMed Central blog.

Viroids: Survivors from the RNA World?

MicrobiologyBytes

Viroid A new review in Annual Review of Microbiology gives an excellent introduction to viroids.

Contents:

  • Introduction: Why The Need For An RNA World?
  • Viroids: Essential Features
  • Discovery of a Subviral World
  • Structure: Small Circular RNAs with Compact Folding
  • Replication: Rolling-Circle Mechanism Catalyzed by Enzymes and Ribozymes
  • Sequence Diversity
  • Viroid-Related Replicons
  • Viroid-Like Satellite RNAs
  • Retroviroid-Like Elements
  • Hepatitis δ virus
  • Why Are Viroids And Viroid-Related Replicons Regarded As Survivors Of The RNA World?
  • Early Speculations on the Origin of Viroids
  • Circular RNAs: Relics of Precellular Evolution?
  • Viroid-Related Replicons
  • Summary

Viroids: Survivors from the RNA World? (2014) Annual Review of Microbiology, 68(1): 395-414. doi: 10.1146/annurev-micro-091313-103416

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The functional role of long non-coding RNAs and epigenetics

Long non-coding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. The post-transcriptional regulation is influenced by these lncRNAs by interfering with the microRNA pathways, involving in diverse cellular processes. The regulation of gene expression by lncRNAs at the epigenetic level, transcriptional and post-transcriptional level have been well known and widely studied. Recent recognition

The functional role of long non-coding RNAs and epigenetics is a post from: lncRNA Blog

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