Hyeshik Chang

Specificity-determining sites (SDS) are the key positions of a protein family that show a specific conservation of amino acids, related to the subfamily members of that family. SDS play crucial role in developing functional variation within the protein family during the course of evolution. Thus, it is important to identify SDS to understand the evolutionary process of diversification of biological functions within a protein family. A wide range of computational tools have been designed to detect such SDS. In this review, we intend to examine the concept of SDS in more details along with the advancements and drawbacks of different computational approaches designed towards successful prediction of SDS. Further, we discussed the algorithms behind the computational approaches developed till date and provide an exhaustive comparison of performance of each method. We also introduce a new ensemble approach, SubSite as another tool to predict SDS through a user-friendly webserver available at www.hpppi.iicb.res.in/subsite.

Jun-Hao Li, Shun Liu, Ling-Ling Zheng, Jie Wu, Wen-Ju Sun, Ze-Lin Wang, Hui Zhou, Liang-Hu Qu and Jian-Hua Yang

The focus on bibliometrics makes papers less useful

Nature 517, 7534 (2015). http://www.nature.com/doifinder/10.1038/517245a

Author: Reinhard Werner

Forcing research to fit the mould of high-impact journals weakens it. Hiring decisions should be based on merit, not impact factor, says Reinhard Werner.

Work–life balance: Lab life with kids

Nature 517, 7534 (2015). doi:10.1038/nj7534-401a

Author: Kendall Powell

Balancing research with raising children takes scheduling skills and organization.

Publication date: March 2015
Source:Trends in Biotechnology, Volume 33, Issue 3
Author(s): João Conde, Natalie Artzi
Only a few years ago pharmaceutical companies were excited about the potential of RNA interference (RNAi). Now, financial volatility and subsequent dissolutions of in-house facilities by pharmaceutical companies have had media channels pronouncing that RNAi therapeutics are dead. However, advances in nanomedicine may now help the vast potential of RNAi therapeutics to be fulfilled.

A model predicts how thousands of disease-linked nucleotide variants affect messenger RNA splicing. [Also see Perspective by Guigó and Valcárcel] Authors: Hui Y. Xiong, Babak Alipanahi, Leo J. Lee, Hannes Bretschneider, Daniele Merico, Ryan K. C. Yuen, Yimin Hua, Serge Gueroussov, Hamed S. Najafabadi, Timothy R. Hughes, Quaid Morris, Yoseph Barash, Adrian R. Krainer, Nebojsa Jojic, Stephen W. Scherer, Benjamin J. Blencowe, Brendan J. Frey

by Athma A. Pai, Jonathan K. Pritchard, Yoav Gilad

It is now well established that noncoding regulatory variants play a central role in the genetics of common diseases and in evolution. However, until recently, we have known little about the mechanisms by which most regulatory variants act. For instance, what types of functional elements in DNA, RNA, or proteins are most often affected by regulatory variants? Which stages of gene regulation are typically altered? How can we predict which variants are most likely to impact regulation in a given cell type? Recent studies, in many cases using quantitative trait loci (QTL)-mapping approaches in cell lines or tissue samples, have provided us with considerable insight into the properties of genetic loci that have regulatory roles. Such studies have uncovered novel biochemical regulatory interactions and led to the identification of previously unrecognized regulatory mechanisms. We have learned that genetic variation is often directly associated with variation in regulatory activities (namely, we can map regulatory QTLs, not just expression QTLs [eQTLs]), and we have taken the first steps towards understanding the causal order of regulatory events (for example, the role of pioneer transcription factors). Yet, in most cases, we still do not know how to interpret overlapping combinations of regulatory interactions, and we are still far from being able to predict how variation in regulatory mechanisms is propagated through a chain of interactions to eventually result in changes in gene expression profiles.

Publication date: 22 January 2015
Source:Molecular Cell, Volume 57, Issue 2
Author(s): Jon Dempersmier , Audrey Sambeat , Olga Gulyaeva , Sarah M. Paul , Carolyn S.S. Hudak , Helena F. Raposo , Hiu-Yee Kwan , Chulho Kang , Roger H.F. Wong , Hei Sook Sul
Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.

Graphical abstract

Teaser

Using high-throughput screening, Dempersmier et al. identify Zfp516 as a cold-inducible regulator of UCP1 transcription. Using both transgenic and knockout mouse models, they show that Zfp516 promotes browning of iWAT to prevent diet-induced obesity and is required for BAT development.

High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles.

Background: The ratio of the rates of non-synonymous and synonymous substitution (d N /d S ) is commonly used to estimate selection in coding sequences. It is often suggested that, all else being equal, d N /d S should be lower in populations with large effective size (N e) due to increased efficacy of purifying selection. As N e is difficult to measure directly, life history traits such as body mass, which is typically negatively associated with population size, have commonly been used as proxies in empirical tests of this hypothesis. However, evidence of whether the expected positive correlation between body mass and d N /d S is consistently observed is conflicting. Results: Employing whole genome sequence data from 48 avian species, we assess the relationship between rates of molecular evolution and life history in birds. We find a negative correlation between d N /d S and body mass, contrary to nearly neutral expectation. This raises the question whether the correlation might be a method artefact. We therefore in turn consider non-stationary base composition, divergence time and saturation as possible explanations, but find no clear patterns. However, in striking contrast to d N /d S , the ratio of radical to conservative amino acid substitutions (K r /K c ) correlates positively with body mass. Conclusions: Our results in principle accord with the notion that non-synonymous substitutions causing radical amino acid changes are more efficiently removed by selection in large populations, consistent with nearly neutral theory. These findings have implications for the use of d N /d S and suggest that caution is warranted when drawing conclusions about lineage-specific modes of protein evolution using this metric.

Gene translation modeling and prediction is a fundamental problem that has numerous biomedical implementations. In this work we present a novel, user-friendly tool/index for calculating the mean of the typical decoding rates that enables predicting translation elongation efficiency of protein coding genes for different tissue types, developmental stages, and experimental conditions. The suggested translation efficiency index is based on the analysis of the organism’s ribosome profiling data. This index could be used for example to predict changes in translation elongation efficiency of lowly expressed genes that usually have relatively low and/or biased ribosomal densities and protein levels measurements, or can be used for example for predicting translation efficiency of new genetically engineered genes. We demonstrate the usability of this index via the analysis of six organisms in different tissues and developmental stages. Distributable cross platform application and guideline are available for download at: http://www.cs.tau.ac.il/~tamirtul/MTDR/MTDR_Install.html

Related Articles

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis.

Cell Cycle. 2014 Jun 1;13(11):1671-6

Authors: Fukunaga R, Zamore PD

Abstract
The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5'-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.

PMID: 24787225 [PubMed - indexed for MEDLINE]

Related Articles

Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions.

Genome Biol. 2014;15(12):537

Authors: Kelley DR, Hendrickson DG, Tenen D, Rinn JL

Abstract
BACKGROUND: Transposable elements (TEs) have significantly influenced the evolution of transcriptional regulatory networks in the human genome. Post-transcriptional regulation of human genes by TE-derived sequences has been observed in specific contexts, but has yet to be systematically and comprehensively investigated. Here, we study a collection of 75 CLIP-Seq experiments mapping the RNA binding sites for a diverse set of 51 human proteins to explore the role of TEs in post-transcriptional regulation of human mRNAs and lncRNAs via RNA-protein interactions.
RESULTS: We detect widespread interactions between RNA binding proteins (RBPs) and many families of TE-derived sequence in the CLIP-Seq data. Further, alignment coverage peaks on specific positions of the TE consensus sequences, illuminating a diversity of TE-specific RBP binding motifs. Evidence of binding and conservation of these motifs in the nonrepetitive transcriptome suggests that TEs have generally appropriated existing sequence preferences of the RBPs. Depletion assays for numerous RBPs show that TE-derived binding sites affect transcript abundance and splicing similarly to nonrepetitive sites. However, in a few cases the effect of RBP binding depends on the specific TE family bound; for example, the ubiquitously expressed RBP HuR confers transcript stability unless bound to an Alu element.
CONCLUSIONS: Our meta-analysis suggests a widespread role for TEs in shaping RNA-protein regulatory networks in the human genome.

PMID: 25572935 [PubMed - in process]

Single-Cell Transcriptome Analysis Reveals Dynamic Changes in lncRNA Expression during Reprogramming.

Cell Stem Cell. 2015 Jan 8;16(1):88-101

Authors: Kim DH, Marinov GK, Pepke S, Singer ZS, He P, Williams B, Schroth GP, Elowitz MB, Wold BJ

Abstract
Cellular reprogramming highlights the epigenetic plasticity of the somatic cell state. Long noncoding RNAs (lncRNAs) have emerging roles in epigenetic regulation, but their potential functions in reprogramming cell fate have been largely unexplored. We used single-cell RNA sequencing to characterize the expression patterns of over 16,000 genes, including 437 lncRNAs, during defined stages of reprogramming to pluripotency. Self-organizing maps (SOMs) were used as an intuitive way to structure and interrogate transcriptome data at the single-cell level. Early molecular events during reprogramming involved the activation of Ras signaling pathways, along with hundreds of lncRNAs. Loss-of-function studies showed that activated lncRNAs can repress lineage-specific genes, while lncRNAs activated in multiple reprogramming cell types can regulate metabolic gene expression. Our findings demonstrate that reprogramming cells activate defined sets of functionally relevant lncRNAs and provide a resource to further investigate how dynamic changes in the transcriptome reprogram cell state.

PMID: 25575081 [PubMed - in process]

Detection of post-transcriptional RNA editing events.

Methods Mol Biol. 2015;1269:189-205

Authors: Picardi E, D'Erchia AM, Gallo A, Pesole G

Abstract
The advent of deep sequencing technologies has greatly improved the study of complex eukaryotic genomes and transcriptomes, providing the unique opportunity to investigate posttranscriptional molecular mechanisms as alternative splicing and RNA editing at single base-pair resolution. RNA editing by adenosine deamination (A-to-I) is widespread in humans and can lead to a variety of biological effects depending on the RNA type or the RNA region involved in the editing modification.Hereafter, we describe an easy and reproducible computational protocol for the identification of candidate RNA editing sites in human using deep transcriptome (RNA-Seq) and genome (DNA-Seq) sequencing data.

PMID: 25577380 [PubMed - in process]

Using EMOTE to Map the Exact 5'-Ends of Processed RNA on a Transcriptome-Wide Scale.

Methods Mol Biol. 2015;1259:69-85

Authors: Redder P

Abstract
The presence or absence of structure in an RNA is often crucial to its function. This is evident for highly structured RNAs such as rRNA, tRNA, or riboswitches, but it is also the case for many mRNAs, where secondary structures in the 5' or 3' UTR can determine the efficiency of translation or the half-life of the RNA. There are paths to modify such secondary structures, (1) by the action of a helicase that allows an alternative RNA structure to form, (2) by the formation of a duplex with another RNA, or (3) by cleavage of the RNA in a way that favors a different secondary structure. None of the three exclude the others, and in vivo it is common that two or all three work together to remodel an RNA to the desired form. However, while the first two solutions can be reversible, the cleavage of RNA is final, and there is no chance to go back. In this chapter, a method for tracking the 5' end created by RNA processing on a transcriptome-wide scale is presented. The Exact Mapping Of Transcriptome Ends (EMOTE) allows the large-scale identification of mono-phosphorylated RNA 5'-ends and provides the exact processing sites.

PMID: 25579580 [PubMed - in process]

Publication date: 13 January 2015
Source:Cell Reports, Volume 10, Issue 2
Author(s): Michal Lubas , Peter Refsing Andersen , Aleks Schein , Andrzej Dziembowski , Grzegorz Kudla , Torben Heick Jensen
The RNA exosome complex constitutes the major nuclear eukaryotic 3′-5′ exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT) complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and 3′-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3′ ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3′-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3′ ends.

Graphical abstract

Teaser

The nuclear RNA exosome is directed to many of its nucleoplasmic substrates by the nuclear exosome targeting (NEXT) complex. Lubas et al. now use iCLIP to follow NEXT targeting to RNA via its RNA binding component RBM7. Association of RBM7 with RNAPII-derived RNAs enables degradation upon emergence of unprotected RNA 3′ ends. Thus, the NEXT complex defines an early exosome targeting pathway acting on newly synthesized RNA, including snoRNAs embedded in pre-mRNA introns.

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

Human 2'-5' oligoadenylate synthetase-1 (OAS1) is central in innate immune system detection of cytoplasmic double-stranded RNA (dsRNA) and promotion of host antiviral responses. However, the molecular signatures that promote OAS1 activation are currently poorly defined. We show that the 3'-end polyuridine sequence of viral and cellular RNA polymerase III non-coding transcripts is critical for their optimal activation of OAS1. Potentiation of OAS1 activity was also observed with a model dsRNA duplex containing an OAS1 activation consensus sequence. We determined that the effect is attributable to a single appended 3'-end residue, is dependent upon its single-stranded nature with strong preference for pyrimidine residues and is mediated by a highly conserved OAS1 residue adjacent to the dsRNA binding surface. These findings represent discovery of a novel signature for OAS1 activation, the 3'-single-stranded pyrimidine (3'-ssPy) motif, with potential functional implications for OAS1 activity in its antiviral and other anti-proliferative roles.

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.

Related Articles

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.

Proc Natl Acad Sci U S A. 2015 Jan 7;

Authors: La Rocca G, Olejniczak SH, González AJ, Briskin D, Vidigal JA, Spraggon L, DeMatteo RG, Radler MR, Lindsten T, Ventura A, Tuschl T, Leslie CS, Thompson CB

Abstract
MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3' untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase-RAC-alpha serine/threonine-protein kinase-mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

PMID: 25568082 [PubMed - as supplied by publisher]

MicroRNAs bind to and regulate the abundance and activity of target messenger RNA through sequestration, enhanced degradation, and suppression of translation. Although miRNA have a predominantly negative effect on the target protein concentration, several reports have demonstrated a positive effect of miRNA, i.e., increase in target protein concentration on miRNA overexpression and decrease in target concentration on miRNA repression. miRNA–target pair-specific effects such as protection of mRNA degradation owing to miRNA binding can explain some of these effects. However, considering such pairs in isolation might be an oversimplification of the RNA biology, as it is known that one miRNA interacts with several targets, and conversely target mRNA are subject to regulation by several miRNAs. We formulate a mathematical model of this combinatorial regulation of targets by multiple miRNA. Through mathematical analysis and numerical simulations of this model, we show that miRNA that individually have a negative effect on their targets may exhibit an apparently positive net effect when the concentration of one miRNA is experimentally perturbed by repression/overexpression in such a multi-miRNA multitarget situation. We show that this apparent unexpected effect is due to competition and will not be observed when miRNA interact noncompetitively with the target mRNA. This result suggests that some of the observed unusual positive effects of miRNA may be due to the combinatorial complexity of the system rather than due to any inherently unusual positive effect of the miRNA on its target.

Nature Biotechnology 33, 107 (2015). doi:10.1038/nbt.3116

Authors: Kathy L Nugent & Lori Lindburg

As the biotech industry grows and shifts within an increasingly global economy, so does its need for talent that spans discovery through commercialization.

Nature Structural & Molecular Biology 22, 89 (2015). doi:10.1038/nsmb.2934

Authors: Maximiliano M Portal, Valeria Pavet, Cathie Erb & Hinrich Gronemeyer

Nature Structural & Molecular Biology 22, 5 (2015). doi:10.1038/nsmb.2942

Authors: John S Mattick & John L Rinn

Nature Structural & Molecular Biology 22, 29 (2015). doi:10.1038/nsmb.2921

Authors: Ci Chu, Robert C Spitale & Howard Y Chang

Nature Structural & Molecular Biology 22, 1 (2015). doi:10.1038/nsmb.2952

The long-held view that the primary role of RNA is to code for proteins has been severely undermined. This Focus explores the remarkable functional diversity of RNA in light of recent breakthroughs in noncoding-RNA biology.

Publication date: 5 January 2015
Source:Current Biology, Volume 25, Issue 1
Author(s): Patrick Bateson
Playful play is undoubtedly fun. Even so, many people think, incorrectly, that as they get older, they are no longer capable of such frivolous activity. They should heed George Bernard Shaw’s advice: “We don’t stop playing because we grow old, we grow old because we stop playing.” The motivation to be playful comes from within. No external bribes are needed. In fact attempting to encourage such activity with food or money is likely to be counterproductive.

Teaser

In this Primer, Patrick Bateson discusses the relationship between playfulness and creativity.

Over the last two decades, advances in experimental and computational technologies have greatly facilitated genomic research. Next-generation sequencing technologies have made de novo sequencing of large genomes affordable, and powerful computational approaches have enabled accurate annotations of genomic DNA sequences. Charting functional regions in genomes must account for not only the coding sequences, but also noncoding RNAs, repetitive elements, chromatin states, epigenetic modifications, and gene regulatory elements. A mix of comparative genomics, high-throughput biological experiments, and machine learning approaches has played a major role in this truly global effort. Here we describe some of these approaches and provide an account of our current understanding of the complex landscape of the human genome. We also present overviews of different publicly available, large-scale experimental data sets and computational tools, which we hope will prove beneficial for researchers working with large and complex genomes.