The RNA exosome affects iron response and sensitivity to oxidative stress. Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets. + Author Affiliations ↵5 These authors contributed equally to this work. Abstract Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. Received February 24, 2014. A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA. Abstract Processing of primary microRNA (pri-miRNA) stem–loops by the Drosha–DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear.
Two prevalent but seemingly conflicting models propose that Drosha–DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Received January 24, 2014. The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA. + Author Affiliations Abstract SBP2 is a pivotal protein component in selenoprotein synthesis.
It binds the SECIS stem–loop in the 3′ UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA.
Received February 20, 2014. The RIDL hypothesis: transposable elements as functional domains of long noncoding RNAs. + Author Affiliations Abstract Our genome contains tens of thousands of long noncoding RNAs (lncRNAs), many of which are likely to have genetic regulatory functions. It has been proposed that lncRNA are organized into combinations of discrete functional domains, but the nature of these and their identification remain elusive. One class of sequence elements that is enriched in lncRNA is represented by transposable elements (TEs), repetitive mobile genetic sequences that have contributed widely to genome evolution through a process termed exaptation.
Here, we link these two concepts by proposing that exonic TEs act as RNA domains that are essential for lncRNA function. We term such elements Repeat Insertion Domains of LncRNAs (RIDLs). Received January 28, 2014. The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit. RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction.
+ Author Affiliations ↵3 These authors contributed equally to this work. Abstract Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID ( an RNA-associated (RNA–RNA/RNA–protein) interaction database.
RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Received February 10, 2014. Elastic network models capture the motions apparent within ensembles of RNA structures. + Author Affiliations + Author Notes ↵3 Present address: Division of Biomedical Statistics and Informatics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Abstract The role of structure and dynamics in mechanisms for RNA becomes increasingly important.
Computational approaches using simple dynamics models have been successful at predicting the motions of proteins and are often applied to ribonucleo-protein complexes but have not been thoroughly tested for well-packed nucleic acid structures. In order to characterize a true set of motions, we investigate the apparent motions from 16 ensembles of experimentally determined RNA structures.
These indicate a relatively limited set of motions that are captured by a small set of principal components (PCs). Received July 12, 2013. RNA recognition by a human antibody against brain cytoplasmic 200 RNA. + Author Affiliations Abstract Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands.
Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Received June 25, 2013. Predicting structure and stability for RNA complexes with intermolecular loop–loop base-pairing. + Author Affiliations + Author Notes ↵3 Present address: The Genome Institute, Washington University School of Medicine in St. Louis, St. Louis, MO 63108, USA ↵2 These authors contributed equally to this work. Abstract RNA loop–loop interactions are essential for genomic RNA dimerization and regulation of gene expression.
Received December 15, 2013. Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani. Structural dynamics of a single-stranded RNA–helix junction using NMR. + Author Affiliations Abstract Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, 13C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A6-tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end.
Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Received November 30, 2013. Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops. Abstract Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined.
The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair.
Received October 30, 2013. RNA secondary structure modeling at consistent high accuracy using differential SHAPE. Abstract RNA secondary structure modeling is a challenging problem, and recent successes have raised the standards for accuracy, consistency, and tractability. Large increases in accuracy have been achieved by including data on reactivity toward chemical probes: Incorporation of 1M7 SHAPE reactivity data into an mfold-class algorithm results in median accuracies for base pair prediction that exceed 90%.
However, a few RNA structures are modeled with significantly lower accuracy. Here, we show that incorporating differential reactivities from the NMIA and 1M6 reagents—which detect noncanonical and tertiary interactions—into prediction algorithms results in highly accurate secondary structure models for RNAs that were previously shown to be difficult to model. For these RNAs, 93% of accepted canonical base pairs were recovered in SHAPE-directed models. Discrepancies between accepted and modeled structures were small and appear to reflect genuine structural differences. A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells. + Author Affiliations Abstract Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome.
Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells.
Analysis of three independent clones showed a total loss of miR-21 expression. Received August 15, 2013. Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex. In vitro characterization of 6S RNA release-defective mutants uncovers features of pRNA-dependent release from RNA polymerase in E. coli. MiRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure. + Author Affiliations Abstract microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Received December 12, 2013. Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting.
A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length. Prevalent RNA recognition motif duplication in the human genome. + Author Affiliations Abstract The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein.
We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. Received December 20, 2013. A versatile assay for RNA-binding proteins in living cells. + Author Affiliations ↵3 These authors contributed equally to this work. Abstract RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. Received November 19, 2013. Mod-seq: high-throughput sequencing for chemical probing of RNA structure.
RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template. Distinct functional classes of ram mutations in 16S rRNA. Principles of ion recognition in RNA: insights from the group II intron structures. The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA. Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs—commonalities and differences. The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome. Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions.
Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host. Tissue-specific gene silencing monitored in circulating RNA. A convenient system for highly specific and sensitive detection of miRNA expression. RNA detection in situ with FISH-STICs. Identification of expressed and conserved human noncoding RNAs. Bacterial RNA motif in the 5′ UTR of rpsF interacts with an S6:S18 complex. Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site. An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Structural and thermodynamic signatures that define pseudotriloop RNA hairpins. Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions.
SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization. Evolutionary and ontogenetic changes in RNA editing in human, chimpanzee, and macaque brains. Rational design of microRNA–siRNA chimeras for multifunctional target suppression. LigandRNA: computational predictor of RNA–ligand interactions. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.
Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance. Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6. Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules. Specific features of telomerase RNA from Hansenula polymorpha. Imp3 unfolds stem structures in pre-rRNA and U3 snoRNA to form a duplex essential for small subunit processing.
Modeling the binding specificity of the RNA-binding protein GLD-1 suggests a function of coding region–located sites in translational repression. Ribosome biogenesis requires a highly diverged XRN family 5′→3′ exoribonuclease for rRNA processing in Trypanosoma brucei. Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging. 2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.
Nanometer scale pores similar in size to the entrance of the ribosomal exit cavity are a common feature of large RNAs. Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription. RNase III is required for localization to the nucleoid of the 5′ pre-rRNA leader and for optimal induction of rRNA synthesis in E. coli. RNA structural motif recognition based on least-squares distance. 2-Selenouridine triphosphate synthesis and Se-RNA transcription. The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal. The tedious task of finding homologous noncoding RNA genes. The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease.
Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA. Minotaur is critical for primary piRNA biogenesis. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element. A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes. ShortStack: Comprehensive annotation and quantification of small RNA genes. RNA. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging. Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme. Crystal structure of Schmallenberg orthobunyavirus nucleoprotein–RNA complex reveals a novel RNA sequestration mechanism.
Crystal structure of Schmallenberg orthobunyavirus nucleoprotein–RNA complex reveals a novel RNA sequestration mechanism. A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes. Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging. Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly. The plasticity of a structural motif in RNA: Structural polymorphism of a kink turn as a function of its environment.