Hyeshik Chang

PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins.

Methods Enzymol. 2014;539:113-61

Authors: Spitzer J, Hafner M, Landthaler M, Ascano M, Farazi T, Wardle G, Nusbaum J, Khorshid M, Burger L, Zavolan M, Tuschl T

Abstract
We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010). Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.

PMID: 24581442 [PubMed - in process]

by Yu-Yuan Hsiao, Woei-Horng Fang, Chia-Chia Lee, Yi-Ping Chen, Hanna S. Yuan

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3′ end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3′ end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3′ overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V–dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3′-end processing.

Motivation: The majority of next-generation sequencing technologies effectively sample small amounts of DNA or RNA that are amplified (i.e. copied) before sequencing. The amplification process is not perfect, leading to extreme bias in sequenced read counts. We present a novel procedure to account for amplification bias and demonstrate its effectiveness in mitigating gene length dependence when estimating true gene expression.

Results: We tested the proposed method on simulated and real data. Simulations indicated that our method captures true gene expression more effectively than classic censoring-based approaches and leads to power gains in differential expression testing, particularly for shorter genes with high transcription rates. We applied our method to an unreplicated Arabidopsis RNA-seq dataset resulting in disparate gene ontologies arising from gene set enrichment analyses.

Availability and implementation: R code to perform the RASTA procedures is freely available on the web at www.stat.purdue.edu/~doerge/.

Contact: doerge@purdue.edu

Nature Genetics 46, 219 (2014). doi:10.1038/ng.2909

Author: Brooke LaFlamme

When bound to the 3' poly(A) tail of mRNA, poly(A)-binding protein (PABP) modulates mRNA translation and stability through its association with various proteins. By visualizing individual PABP molecules in real time, we found that PABP, containing four RNA recognition motifs (RRMs), adopts a conformation on poly(A) binding in which RRM1 is in proximity to RRM4. This conformational change is due to the bending of the region between RRM2 and RRM3. PABP-interacting protein 2 actively disrupts the bent structure of PABP to the extended structure, resulting in the inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration of PABP induced by interactions with various effector molecules, such as poly(A) and PABP-interacting protein 2, play pivotal roles in its function.

Chemical landscape of natural RNA species is decorated with the large number of modified nucleosides. Some of those could easily be detected by reverse transcription, while others permit only high-performance liquid chromatography or mass-spectrometry detection. Presence of m⁶A nucleoside at a particular position of long RNA molecule is challenging to observe. Here we report an easy and high-throughput method for detection of m⁶A nucleosides in RNA based on high-resolution melting analysis. The method relies on the previous knowledge of the modified nucleoside position at a particular place of RNA and allows rapid screening for conditions or genes necessary for formation of that modification.

Nature Protocols 9, 711 (2014). doi:10.1038/nprot.2014.043

Authors: Aleksandra Helwak & David Tollervey

RNA-RNA interactions have critical roles in many cellular processes, but studying them is difficult and laborious. Here we describe an experimental procedure, termed cross-linking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein

Nature Methods 11, 301 (2014). doi:10.1038/nmeth.2806

Authors: Ching-Yun Chang, Eduard Sabidó, Ruedi Aebersold & Olga Vitek

Targeted proteomics is a method of choice for accurate and high-throughput quantification of predefined sets of proteins. Many workflows use isotope-labeled reference peptides for every target protein, which is time consuming and costly. We report a statistical approach for quantifying full protein panels with a reduced set of reference peptides. This label-sparse approach achieves accurate quantification while reducing experimental cost and time. It is implemented in the software tool SparseQuant.

Nature Methods 11, 215 (2014). doi:10.1038/nmeth.2858

Authors: Martin Krzywinski & Naomi Altman

Robustly comparing pairs of independent or related samples requires different approaches to the t-test.

Nature Methods 11, 235 (2014). doi:10.1038/nmeth.2852

Authors: Ulrike Endesfelder & Mike Heilemann

Single-molecule super-resolution techniques emerged only several years ago but have revolutionized fluorescence microscopy of cellular structures. We discuss some key principles of these techniques, point out pitfalls, highlight recent developments and identify opportunities for the future.

Nicholas R. Guydosh, Rachel Green. Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vi....

Agata L. Starosta, Daniel N. Wilson. In eukaryotes, Dom34 is involved in the rescue of ribosomes that stall on mRNAs during protein synthesis. Using ribosome profiling, Guydosh and Green reveal that, in addition to rescue of ribosome....

Here we describe how to prepare nucleoplasmic extracts from suspension cultures of Drosophila S2 (Schneider Line 2) cells. Harvested cells are washed in phosphate-buffered saline supplemented with MgCl₂, resuspended in hypotonic buffer, and homogenized. Nuclei are isolated by centrifugation and then sonicated. The nuclear sonicate is placed on a 30% sucrose cushion and sedimented. The soluble nuclear ribonucleoprotein (RNP) complexes remain in the supernatant and the nuclear membrane fragments and chromatin pellet through the sucrose cushion.

The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible...

Advances in high-throughput transcriptome analyses have revealed hundreds of antisense RNAs (asRNAs) for many bacteria, although few have been characterized, and the number of functional asRNAs remains unknown. We have developed a genome-wide high-throughput method to identify functional asRNAs in vivo. Most mechanisms of gene regulation via asRNAs require an...

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ~1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

It is well known that metal ions play essential roles in RNA folding and catalysis. This study examines in detail metal ions bound to specific regions of a catalytic group II intron. These analyses based on crystallographic data provide new insight into how metal ions are localized in complex folded RNA structures.

In this paper, the authors present a nonradioactive Northern blotting method for the detection of miRNAs. This protocol, which takes advantage of the previously developed splinted ligation approach for detecting small RNAs, also includes cross-linking with EDC, which improves capture of small RNAs and the use of a DIG-labeled probe to eliminate the need for radioactivity.

by Andrew G. Hoss, Vinay K. Kartha, Xianjun Dong, Jeanne C. Latourelle, Alexandra Dumitriu, Tiffany C. Hadzi, Marcy E. MacDonald, James F. Gusella, Schahram Akbarian, Jiang-Fan Chen, Zhiping Weng, Richard H. Myers

Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value

by Patrick Linder, Sylvain Lemeille, Peter Redder

RNA decay and maturation have in recent years been recognised as major regulatory mechanisms in bacteria. In contrast to Escherichia coli, the Firmicute (Gram-positive) bacteria often do not encode the well-studied endonuclease RNase E, but instead rely on the endonucleases RNase Y, RNase J1 and RNase J2, of which the latter two have additionally been shown to have 5′ to 3′ exonucleolytic activity. We have previously demonstrated that these RNases could be deleted individually in the pathogenic Firmicute Staphylococcus aureus; however, we here present that, outside a narrow permissive window of growth conditions, deleting one or both of the RNase J genes presents serious difficulties for the cell. Moreover, an active site mutant of RNase J1 behaved like a deletion, whereas no phenotypes were detected for the RNase J2 active site mutant. Furthermore, in order to study the in vivo enzymatic activity of RNase J1 and J2, a method was developed to map the exact 5′-ends of mature and processed RNA, on a global scale. An enrichment of 5′ RNA ends could be seen in the RNase J mutants, suggesting that their exonucleolytic activity is crucial for normal degradation of bulk RNA. Using the data to examine specific RNAs, we demonstrated that RNase J activity is needed for correct 5′ maturation of both the 16S rRNA and the RNase P ribozyme, and can also inactivate the latter, possibly as quality control. Additional examples show that RNase J perform initial cleavages, apparently competing with ribosomes for access to mRNAs. The novel 5′ mapping assay offers an exceptionally detailed view of RNase activity, and reveals that the roles of RNase J proteins are diverse, ranging from maturation and post-transcriptional regulation to degradation.

Highly Multiplexed Subcellular RNA Sequencing in Situ.

Science. 2014 Feb 27;

Authors: Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SS, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J, Church GM

Abstract
Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8742 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

PMID: 24578530 [PubMed - as supplied by publisher]

Related Articles

Biogenesis of intronic miRNAs located in clusters by independent transcription and alternative splicing.

RNA. 2014 Jan;20(1):76-87

Authors: Ramalingam P, Palanichamy JK, Singh A, Das P, Bhagat M, Kassab MA, Sinha S, Chattopadhyay P

Abstract
miRNAs are generally classified as "intergenic" or "intronic" based upon their genomic location. Intergenic miRNAs are known to be transcribed as independent transcription units, while intronic miRNAs are believed to be processed from the introns of their hosting transcription units and hence share common regulatory mechanisms and expression patterns with its host gene. Recent reports in the literature suggest that some intronic miRNAs, which do not show concordance in expression with their respective host genes, might be transcribed and regulated as independent transcription units. However, there is no direct evidence for the existence of independently transcribed intronic miRNA in humans to date. We have characterized the full-length primary transcripts (pri-miRNAs) of three human intronic miRNAs-miR 106b, miR 93, and miR 24-1-by RNA ligase-mediated RACE and show that human intronic miRNA can indeed be transcribed as independent transcription units. Also, clustered miRNAs are generally believed to arise from a common primary transcript and are expected to have similar expression profiles. However, we have identified several novel alternatively spliced transcripts by RT-PCR, each of which harbors a single pre-miRNA from a cluster of closely located intronic miRNAs. We show that these transcripts represent unique pri-miRNAs for each of these clustered miRNAs. We also report the identification of conserved splice acceptor signals which are responsible for maturation of these novel splice variants. Our results suggest that alternative splicing might play a role in uncoupling the expression of clustered miRNAs from each other, which otherwise are generally believed to be co-transcribed and co-expressed.

PMID: 24226766 [PubMed - indexed for MEDLINE]

Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions.

Mol Syst Biol. 2014;10(2):719

Authors: Gupta I, Clauder-Münster S, Klaus B, Järvelin AI, Aiyar RS, Benes V, Wilkening S, Huber W, Pelechano V, Steinmetz LM

Abstract
Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3' untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3' UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

PMID: 24569168 [PubMed - in process]

Related Articles

Detection of 3'-end RNA uridylation with a protein nanopore.

ACS Nano. 2014 Feb 25;8(2):1364-74

Authors: Clamer M, Höfler L, Mikhailova E, Viero G, Bayley H

Abstract
Post-transcriptional modifications of the 3'-ends of RNA molecules have a profound impact on their stability and processing in the cell. Uridylation, the addition of uridines to 3'-ends, has recently been found to be an important regulatory signal to stabilize the tagged molecules or to direct them toward degradation. Simple and cost-effective methods for the detection of this post-transcriptional modification are not yet available. Here, we demonstrate the selective and transient binding of 3'-uridylated ssRNAs inside the β barrel of the staphylococcal α-hemolysin (αHL) nanopore and investigate the molecular basis of uridine recognition by the pore. We show the discrimination of 3'-oligouridine tails on the basis of their lengths and propose the αHL nanopore as a useful sensor for this biologically relevant RNA modification.

PMID: 24369707 [PubMed - in process]

Related Articles

Mitochondrial RNA editing in trypanosomes: Small RNAs in control.

Biochimie. 2014 Jan 17;

Authors: Aphasizhev R, Aphasizheva I

Abstract
Mitochondrial mRNA editing in trypanosomes is a posttranscriptional processing pathway thereby uridine residues (Us) are inserted into, or deleted from, messenger RNA precursors. By correcting frameshifts, introducing start and stop codons, and often adding most of the coding sequence, editing restores open reading frames for mitochondrially-encoded mRNAs. There can be hundreds of editing events in a single pre-mRNA, typically spaced by few nucleotides, with U-insertions outnumbering U-deletions by approximately 10-fold. The mitochondrial genome is composed of ∼50 maxicircles and thousands of minicircles. Catenated maxi- and minicircles are packed into a dense structure called the kinetoplast; maxicircles yield rRNA and mRNA precursors while guide RNAs (gRNAs) are produced predominantly from minicircles, although varying numbers of maxicircle-encoded gRNAs have been identified in kinetoplastids species. Guide RNAs specify positions and the numbers of inserted or deleted Us by hybridizing to pre-mRNA and forming series of mismatches. These 50-60 nucleotide (nt) molecules are 3' uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Editing reactions of mRNA cleavage, U-insertion or deletion, and ligation are catalyzed by the RNA editing core complex (RECC). To function in mitochondrial translation, pre-mRNAs must further undergo post-editing 3' modification by polyadenylation/uridylation. Recent studies revealed a highly compound nature of mRNA editing and polyadenylation complexes and their interactions with the translational machinery. Here we focus on mechanisms of RNA editing and its functional coupling with pre- and post-editing 3' mRNA modification and gRNA maturation pathways.

PMID: 24440637 [PubMed - as supplied by publisher]

Jesse R. Zamudio, Timothy J. Kelly, Phillip A. Sharp. Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic ste....

Ryu-Suke Nozawa, Nick Gilbert. RNA has been proposed to be a component of an underlying nuclear matrix. Hall et al. show that noncoding, repetitive RNAs, some derived from LINE1 elements, stably associate with interphase chromo....

Masaki Mori, Robinson Triboulet, Morvarid Mohseni, Karin Schlegelmilch, Kriti Shrestha, Fernando D. Camargo, Richard I. Gregory. Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly under....

Hyeshik Chang, Jaechul Lim, Minju Ha, V. Narry Kim. Global investigation of the 3′ extremity of mRNA (3′-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequence....