Article
Screens for protein–protein interactions and for drugs that disrupt them typically use in vitro assays which fail to capture the complexity of the cell’s interior. By fixing proteins to distinct cellular locations, Herce et al. demonstrate a fluorescent-three-hybrid approach to probe such interactions in their cellular contexts.
Nature Communications doi: 10.1038/ncomms3660
Authors: Henry D. Herce, Wen Deng, Jonas Helma, Heinrich Leonhardt, M. Cristina Cardoso
T4 RNA ligase 2 (Rnl2) seals 3'-OH/5'-PO4 nicks in double-stranded nucleic acids where the broken strand is RNA. This study analyzed the kinetic mechanism and fidelity of nick sealing by Rnl2–AMP. The data reveal why Rnl2 requires proper positioning of the 3'-terminal ribonucleoside and raise the possibility that nick-sealing ligases could embed mutations during the repair of oxidative damage.
MicroRNA-independent roles of the RNase III enzymes Drosha and Dicer.
Open Biol. 2013;3(10):130144
Authors: Johanson TM, Lew AM, Chong MM
Abstract
The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology.
PMID: 24153005 [PubMed - in process]
MicroRNA or NMD: Why Have Two RNA Silencing Systems?
J Genet Genomics. 2013 Oct 20;40(10):497-513
Authors: Zhang Z, Hu L, Kong X
Abstract
MicroRNA (miRNA)-mediated RNA silencing and nonsense-mediated decay (NMD) are two conserved RNA-level regulatory pathways. Although they are mechanically different, both can regulate target genes by RNA degradation and translational repression. Moreover, studies of individual target genes indicated that these two pathways can be involved in the same processes (e.g., development and stress responses). These facts raise an important question that whether these two systems are cooperative, interchangeable or optimal for regulation of different sorts of genes. We addressed this by comparing miRNA and NMD targets in Arabidopsis thaliana at the genome-wide scale. We find no more overlap in the genes targeted by both systems than expected by chance. Moreover, the sorts of genes or pathways regulated by these systems are categorically different on several cross-correlating fronts. While miRNA targets show enrichment in the process of development, metabolism and transcription, NMD targets are associated with stress responses but otherwise poorly annotated. Validated miRNA targets are more highly expressed, less variably expressed and slower evolving. These differences suggest that the modes of regulation need not be interchangeable. Instead, we suggest that miRNA genes are commonly dose-sensitive and require fine control of levels through weak pull-down by miRNAs. This is consistent with miRNA-regulated genes being more likely to be involved in protein-protein interactions. Many NMD-regulated genes, by contrast, have properties consistent with them being rapid emergency response "fire-fighter" genes. If true, the lack of annotation of NMD targets suggests that we poorly understand the emergencies plants face in the wild.
PMID: 24156916 [PubMed - in process]
Nature advance online publication 27 October 2013. doi:10.1038/nature12651
Authors: Ping Yin, Quanxiu Li, Chuangye Yan, Ying Liu, Junjie Liu, Feng Yu, Zheng Wang, Jiafu Long, Jianhua He, Hong-Wei Wang, Jiawei Wang, Jian-Kang Zhu, Yigong Shi & Nieng Yan
Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI–ATPH and PSAJ–RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5′ or 3′ exonucleases, PPR10 defines the corresponding 5′ and 3′ messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.
Nature Protocols 8, 2281 (2013). doi:10.1038/nprot.2013.143
Authors: F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a
This paper examines A-to-I RNA-editing conservation among infant and mature humans, chimpanzees, and macaques in two brain regions. While RNA-editing patterns are largely conserved within and among species, as well as between brain regions, >15% of sites display human-specific editing patterns. Further, in all three species, RNA-editing levels tend to increase with age.
Genome-wide analysis of polyadenylation signals in Schizosaccharomyces pombe reveals conservation of the ubiquitous AATAAA motif. Heterogeneity of cleavage sites is common, as is alternative polyadenylation. Furthermore, different physiological conditions can have profound effects on poly(A) site usage. These data are accessible in a user-friendly database Pomb(A).
The survival motor neuron (SMN) is known to be involved in the biogenesis of small nuclear RNPs. This study provides evidence that the SMN may also be involved in axonal trafficking of mRNPs. Specifically, in situ hybridization and immuno-fluorescence experiments indicate that many SMN-associated mRNPs are found in neurites and axons, suggesting that SMN deficiency might result in mislocalization of mRNAs required for axonogenesis.
Chromatin remodeling by the small RNA machinery in mammalian cells.
Epigenetics. 2013 Oct 22;9(1)
Authors: Li LC
Abstract
Chromatin states, quite different from changes in DNA sequence, can impact fundamental cellular processes such as determination of cell identity and development of disease. However, how chromatin states are established and regulated remain to be fully elucidated. In several lower eukaryotes, the small RNA machinery comprised of small RNA and its partners, the Argonaute proteins, is known to play important roles in the establishment of heterochromatin and silencing of repetitive sequences. In mammalian cells, however, the nuclear function of the small RNA machinery is largely unknown. Emerging evidence suggests that components of the small RNA pathway interact with chromatin to regulate nuclear events, including gene transcription and alternative splicing. In addition, these endogenous mechanisms are being exploited to target specific genomic loci for manipulation of gene expression and splicing events. In this review, I summarize current understanding of chromatin remodeling by small RNAs in mammalian cells and highlight recent efforts to map genome-wide interactions between RNAi-related factors and chromatin.
PMID: 24149777 [PubMed - as supplied by publisher]
3'-End maturation of histone mRNAs in higher eukaryotes requires U7 snRNA and several factors that are essential for canonical cleavage and polyadenylation. This paper reveals some interesting differences between histone 3'-end formation in Drosophila and mammalian cells. In particular, Ars2 is not essential for Drosophila histone 3'-end processing, and the Drosophila U7 snRNP is associated with several polyadenylation factors in a stable supercomplex.
The authors have designed chimeric small RNAs, termed aiRNAs, which incorporate sequences from both miRNAs and siRNAs. These novel aiRNAs maintain the function of the miRNAs while gaining new functions of gene knockdown in an siRNA-like fashion.
Small Cajal-body-specific RNAs (scaRNAs) guide the post-transcriptional modification of the U1, U2, U4, and U5 spliceosomal RNAs. Seven new scaRNAs from Drosophila were identified by coimmunoprecipitation with the WD-repeat protein WDR79. These scaRNAs were localized in the nuclear Cajal body by in situ hybridization, and their functions as guide RNAs were verified experimentally. Several had unexpected features, including one that modifies U6 snRNA and two with dual guide functions (an snRNA and 28S rRNA) that imply a previously unrecognized plasticity of scaRNA-substrate recognition.
In this paper, the authors describe a universal stem–loop primer-based RT-PCR protocol for TaqMan probes which is more cost-efficient and can be used to detect small noncoding RNAs. This new method is efficient for the detection of synthetic siRNAs, double-stranded miRNA mimics, single-stranded synthetic miRNAs, and in vitro transcribed miRNA precursors, as well as for the detection of endogenous bacterial small RNAs.
When converted to cDNA in the course of RNA sequencing, modified RNA nucleotides can produce sequence patterns that are distinguishable from simple base-calling errors. The authors developed a method, HAMR, that can not only locate these modifications transcriptome-wide with single nucleotide resolution, but can also differentiate between different classes of modifications.
Nature Reviews Molecular Cell Biology 14, 699 (2013). doi:10.1038/nrm3679
Authors: Sarah Geisler & Jeff Coller
The increased application of transcriptome-wide profiling approaches has led to an explosion in the number of documented long non-coding RNAs (lncRNAs). While these new and enigmatic players in the complex transcriptional milieu are encoded by a significant proportion of the genome, their functions are mostly
Nature Reviews Molecular Cell Biology 14, 688 (2013). doi:10.1038/nrm3689
Author: Rachel David
This study reports an experimental system that allows the visualization of transcription and mRNA splicing in living human cells, with single-molecule resolution. Using this approach, the authors obtained key insights into splicing dynamics. First, they inserted GFP-tagged versions of the coat protein of bacteriophage MS2
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Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing.
Genome Res. 2013 Jun;23(6):977-87
Authors: Arribere JA, Gilbert WV
Abstract
Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m(7)G-capped mRNA 5' ends with high-throughput sequencing. TL-seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Surprisingly, TL-seq identified transcription initiation sites within 6% of protein-coding regions, and these sites were concentrated near the 5' ends of ORFs. Furthermore, ribosome density analysis showed these truncated mRNAs are translated. Translation-associated TL-seq (TATL-seq), which combines TL-seq with polysome fractionation, enabled annotation of TLs, and simultaneously assayed their function in translation. Using TATL-seq to address relationships between TL features and translation of the downstream ORF, we observed that upstream AUGs (uAUGs), and no other upstream codons, were associated with poor translation and nonsense-mediated mRNA decay (NMD). We also identified hundreds of genes with very short TLs, and demonstrated that short TLs were associated with poor translation initiation at the annotated start codon and increased initiation at downstream AUGs. This frequently resulted in out-of-frame translation and subsequent termination at premature termination codons, culminating in NMD of the transcript. Unlike previous approaches, our technique enabled observation of alternative TL variants for hundreds of genes and revealed significant differences in translation in genes with distinct TL isoforms. TL-seq and TATL-seq are useful tools for annotation and functional characterization of TLs, and can be applied to any eukaryotic system to investigate TL-mediated regulation of gene expression.
PMID: 23580730 [PubMed - indexed for MEDLINE]
Transcription and epigenetics
Nature 502, 7472 (2013). doi:10.1038/502461a
Authors: Alex Eccleston, Francesca Cesari & Magdalena Skipper
Transcriptional programs drive cell identity, dictating the specialized phenotypes of differentiated cells during the development of multicellular organisms. Interplay between signal-transduction pathways, transcription factors and the chromatin packaging of the genome sets the gene expression pattern of a cell, which must be relatively stably maintained
Biochemistry: Icy origins for RNA copying?
Nature 502, 7472 (2013). doi:10.1038/502412d
For the first time, experiments in evolution have produced an RNA molecule that can build other RNA molecules that are longer than itself.Many theories of the origin of life rely on RNA self-replication, but researchers have struggled to make RNA 'enzymes' that can stitch
Universities must inspire students as well as teach
Nature 502, 7472 (2013). http://www.nature.com/doifinder/10.1038/502411a
Author: Rana Dajani
Education in the Arab world must equip students with more than textbook learning as they go forward into an uncertain future, says Rana Dajani.
Nature Chemical Biology 9, 671 (2013). doi:10.1038/nchembio.1334
Authors: Dusica Curanovic, Michael Cohen, Irtisha Singh, Christopher E Slagle, Christina S Leslie & Samie R Jaffrey
Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry–compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation.
It has long been a mystery as to the function(s) of N6-methyladenosine modifications in mRNAs. This paper describes a method to determine both the location and stoicheometry of such modified nucleotides in long RNAs. This method may provide clues regarding potential functions for m6A modifications.
It has been known that Lin28 negatively regulates let-7 production by recruiting 3'-terminal uridylyl transferases. As a result, the processing of uridylated pre-let-7 by Dicer is blocked. The identity of the RNase that acts to degrade uridylated pre-let-7 was not known until recently. In this paper, the authors report the identification of mammalian DIS3L2 as an oligo(U)-binding exonuclease that specifically targets uridylated let-7 miRNA precursors in vivo. These findings complement a recent report which identified Dis3l2 as the relevant mouse exonuclease. DIS3L2 has 3'–5' exonuclease activity, is predominantly cytoplasmic and, unlike other mammalian DIS3 orthologs, is not stably associated with the RNA exosome complex.
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Predicting RNA structure: advances and limitations.
Methods Mol Biol. 2014;1086:1-19
Authors: Hofacker IL, Lorenz R
Abstract
RNA secondary structures can be predicted using efficient algorithms. A widely used software package implementing a large number of computational methods is the ViennaRNA Package. This chapter describes how to use programs from the ViennaRNA Package to perform common tasks such as prediction of minimum free-energy structures, suboptimal structures, or base pairing probabilities, and generating secondary structure plots with reliability annotation. Moreover, we present recent methods to assess the folding kinetics of an RNA via 2D projections of the energy landscape, identification of local minima and energy barriers, or simulation of RNA folding as a Markov process.
PMID: 24136595 [PubMed - in process]
Identification of Novel MicroRNAs in Primates by Using the Synteny Information and Small RNA Deep Sequencing Data.
Int J Mol Sci. 2013;14(10):20820-20832
Authors: Yuan Z, Liu H, Nie Y, Ding S, Yan M, Tan S, Jin Y, Sun X
Abstract
Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.
PMID: 24135875 [PubMed - as supplied by publisher]
CRISPR interference (CRISPRi) for sequence-specific control of gene expression.
Nat Protoc. 2013 Nov;8(11):2180-2196
Authors: Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS
Abstract
Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
PMID: 24136345 [PubMed - as supplied by publisher]