Hyeshik Chang

Chemical sequencing of RNA relies on the fact that each of the four bases in RNA is susceptible to chemical modification in a different way. In this protocol, end-labeled RNAs are subjected to base-specific chemical modification reactions that make the RNA strand adjacent to the modified base susceptible to cleavage. The chemical modification reaction is base-specific but limited so that not every base in every strand is modified. After cleavage, the resulting set of radioactive fragments is resolved via polyacrylamide gel electrophoresis.

The field of single-cell analysis has greatly benefitted from recent technological advances allowing scientists to study genomes, transcriptomes, proteomes, and metabolomes at the single-cell level. Transcriptomics allows a unique window into cell function and is especially useful for studying global variability among single cells of seemingly the same type. Generating transcriptome data from RNA samples has become increasingly easy and can be done using either microarray or RNA-Seq techniques. RNA isolation is the first step of transcriptomics. Numerous RNA isolation procedures exist and differ with respect to the type and number of cells from which they are capable of isolating RNA. Although it is trivial to isolate RNA from bulk tissue or culture plates, sophisticated methods are required to capture RNA from single cells in a pool of cells or in intact tissue. We describe here the protocols used for isolating the soma of single neurons in cultures and in tissue slices using the pipette capture and the PALM or laser capture microdissection (LCM) approaches, respectively. LCM was developed to isolate cells from tissue sections primarily for pathological tissue analysis. LCM can be used to isolate individual cells or groups of cells from ethanol or paraffin-embedded formalin-fixed tissue sections and dissociated tissue cultures. The soma isolates from either technique can subsequently be used for RNA amplification procedures and transcriptome analysis. These procedures can also be adapted to other cell types in cultures and tissue sections and can be used on neuronal subcellular structures, such as dendrites.

Poisoned primer extension is primarily used to distinguish between RNAs that are nearly identical in sequence but cannot be distinguished by standard primer extension because they are the same size (e.g., edited vs. nonedited transcripts). It is conceptually identical to the standard primer extension reaction but involves the use of a chain-terminating dideoxynucleotide (the "poison") in the presence of the other three nucleotides. A radioactively labeled primer that hybridizes a short-distance downstream from the "changed" region of interest is extended by reverse transcription into this region of sequence variation. The reactions contain three of the four substrates for extension (e.g., dATP, dGTP, and dTTP) and a chain-terminating dideoxynucleotide (e.g., ddCTP). The extension reaction stops when reverse transcriptase adds a chain-terminating dideoxynucleotide to the template (e.g., it will add ddCTP when it encounters a G in the template sequence). RNAs that differ in sequence at that position will yield different-sized extension products that can be resolved on a denaturing gel.

Publication date: 5 January 2015
Source:Current Biology, Volume 25, Issue 1
Author(s): Richard W. Byrne
Fun is functional: play is evolution’s way of making sure animals acquire and perfect valuable skills in circumstances of relative safety. Yet precisely what animals find fun has seldom been examined for what it can potentially reveal about how they represent and think about the world.

Teaser

Fun is functional: play is evolution’s way of making sure animals acquire and perfect valuable skills in circumstances of relative safety. In this Essay, Richard Byrne discusses what animals find fun, and what this may reveal about how they represent and think about the world.

Genome-wide analysis of long noncoding RNA turnover.

Methods Mol Biol. 2015;1262:305-20

Authors: Tani H, Imamachi N, Mizutani R, Imamura K, Kwon Y, Miyazaki S, Maekawa S, Suzuki Y, Akimitsu N

Abstract
Genome-wide analysis for determining RNA turnover is an advanced method in RNA biology that examines the specific half-life of nuclear noncoding RNA (ncRNA). In particular, a pulse-labeling method using uridine analogs enables the determination of RNA stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing. The method is called BrU immunoprecipitation chase assay (BRIC) or BRIC through deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq.

PMID: 25555590 [PubMed - in process]

Nature Reviews Genetics. doi:10.1038/nrg3863

Authors: Daniel Holoch & Danesh Moazed

Publication date: 13 January 2015
Source:Cell Reports, Volume 10, Issue 2
Author(s): Andranik Ivanov , Sebastian Memczak , Emanuel Wyler , Francesca Torti , Hagit T. Porath , Marta R. Orejuela , Michael Piechotta , Erez Y. Levanon , Markus Landthaler , Christoph Dieterich , Nikolaus Rajewsky
Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.

Graphical abstract

Teaser

Ivanov et al. study the biogenesis of circRNAs and reveal its conserved features. Moreover, they show that circRNAs can be predicted based on the genomic sequence of their flanking introns. Finally, they demonstrate that the circRNA formation mechanism depends on the RNA-editing enzyme ADAR.

Regulating Pol III transcription to change Pol II transcriptome.

Cell Cycle. 2014 Dec 1;13(23):3625-3626

Authors: Ichiyanagi K

PMID: 25551358 [PubMed - as supplied by publisher]

Publication date: 5 February 2015
Source:Molecular Cell, Volume 57, Issue 3
Author(s): Ross C. Wilson , Akshay Tambe , Mary Anne Kidwell , Cameron L. Noland , Catherine P. Schneider , Jennifer A. Doudna
RNA-mediated gene silencing in human cells requires the accurate generation of ∼22 nt microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of the human Dicer-TRBP interface, revealing the structural basis of the interaction. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.

Graphical abstract

Teaser

Wilson et al. determine the crystal structure of the interface between microRNA biogenesis proteins Dicer and TRBP. Mutations in this interface prevent recruitment of TRBP to Dicer and reveal how TRBP contributes to the accuracy microRNA processing.

Publication date: 22 January 2015
Source:Molecular Cell, Volume 57, Issue 2
Author(s): Sager J. Gosai , Shawn W. Foley , Dongxue Wang , Ian M. Silverman , Nur Selamoglu , Andrew D.L. Nelson , Mark A. Beilstein , Fevzi Daldal , Roger B. Deal , Brian D. Gregory
Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Graphical abstract

Teaser

RNA secondary structure and RNA-protein interactions regulate all aspects of a transcript’s life cycle. Using a ribonuclease-mediated protein-footprinting approach, Gosai et al. provide a simultaneous genome-wide view of RNA-protein interaction sites and RNA secondary structure in the Arabidopsis nucleus.

Stalled protein translation results in 80S ribosome– and messenger RNA–free amino acid addition to truncated proteins. Authors: Peter S. Shen, Joseph Park, Yidan Qin, Xueming Li, Krishna Parsawar, Matthew H. Larson, James Cox, Yifan Cheng, Alan M. Lambowitz, Jonathan S. Weissman, Onn Brandman, Adam Frost

Nature Methods 12, 5 (2015). doi:10.1038/nmeth.3224

Authors: Naomi Altman & Martin Krzywinski

Nature Methods 12, 7 (2015). doi:10.1038/nmeth.3213

Authors: Jianyi Yang, Renxiang Yan, Ambrish Roy, Dong Xu, Jonathan Poisson & Yang Zhang

High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.

Mar Genomics. 2014 Dec 26;

Authors: Asai S, Ianora A, Lauritano C, Lindeque PK, Carotenuto Y

Abstract
Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.

PMID: 25546577 [PubMed - as supplied by publisher]

Human long noncoding RNAs are substantially less folded than messenger RNAs.

Mol Biol Evol. 2014 Dec 23;

Authors: Yang J, Zhang J

Abstract
Long noncoding RNAs (lncRNAs) do not code for proteins but function as RNAs. Because the functions of an RNA rely on either its sequence or secondary structure, lncRNAs should be folded at least as strongly as messenger RNAs (mRNAs), which serve as messengers for translation and are generally thought to lack secondary structure-dependent RNA-level functions. Contrary to this prediction, analysis of genome-wide experimental data of human RNA folding reveals that lncRNAs are substantially less folded than mRNAs even after the control of expression level and GC%, although both lncRNAs and mRNAs are more strongly folded than expected by chance. In contrast to mRNAs, lncRNAs show neither the positive correlation between folding strength and expression level nor the negative correlation between folding strength and evolutionary rate. These and other results support that, while RNA folding undoubtedly plays a role in RNA biology, it is also important in translation and/or protein biology.

PMID: 25540450 [PubMed - as supplied by publisher]

Dissecting neural differentiation regulatory networks through epigenetic footprinting.

Nature. 2014 Dec 24;

Authors: Ziller MJ, Edri R, Yaffe Y, Donaghey J, Pop R, Mallard W, Issner R, Gifford CA, Goren A, Xing J, Gu H, Cacchiarelli D, Tsankov AM, Epstein C, Rinn JL, Mikkelsen TS, Kohlbacher O, Gnirke A, Bernstein BE, Elkabetz Y, Meissner A

Abstract
Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

PMID: 25533951 [PubMed - as supplied by publisher]

Publication date: 8 January 2015
Source:Molecular Cell, Volume 57, Issue 1
Author(s): Jiayu Wen , Hong Duan , Fernando Bejarano , Katsutomo Okamura , Lacramioara Fabian , Julie A. Brill , Diane Bortolamiol-Becet , Raquel Martin , J. Graham Ruby , Eric C. Lai
Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-β in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.

Graphical abstract

Teaser

While the biochemical mechanism and technical applications of RNAi are well-established, still little is known about its endogenous utility. Here, Wen et al. show that the Drosophila hpRNA/RNAi pathway mediates adaptive regulation of specific target genes in the testis and that this is required for spermatogenesis and normal male fertility.

Publication date: 6 January 2015
Source:Cell Reports, Volume 10, Issue 1
Author(s): Stefan Starke , Isabelle Jost , Oliver Rossbach , Tim Schneider , Silke Schreiner , Lee-Hsueh Hung , Albrecht Bindereif
Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode.

Graphical abstract

Teaser

Starke et al. conclusively demonstrate a circular configuration of two predicted circular RNAs and analyze the sequence requirements for exon circularization. Canonical splice signals are required for circularization, which together with splice inhibitor assays indicates that the canonical spliceosome machinery functions in the biogenesis of circular RNAs.

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.

Nat Chem Biol. 2014 Dec 22;

Authors: Imig J, Brunschweiger A, Brümmer A, Guennewig B, Mittal N, Kishore S, Tsikrika P, Gerber AP, Zavolan M, Hall J

Abstract
Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

PMID: 25531890 [PubMed - as supplied by publisher]

by Lilian T. Lamech, Anna L. Mallam, Alan M. Lambowitz

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation...

Nature Reviews Molecular Cell Biology 16, 3 (2015). doi:10.1038/nrm3928

Author: Eytan Zlotorynski

Measuring translation initiation rates is difficult. Gao et al. developed quantitative translation initiation sequencing (QTI-seq), a technique that captures translation initiation sites (TISs) at single-nucleotide resolution in cells and solid tissues, based on dissociating elongating ribosomes from transcripts while preserving the initiating ribosomes. The

Nature advance online publication 22 December 2014. doi:10.1038/nature14020

Authors: Hana Cahová, Marie-Luise Winz, Katharina Höfer, Gabriele Nübel & Andres Jäschke

A distinctive feature of prokaryotic gene expression is the absence of 5′-capped RNA. In eukaryotes, 5′,5′-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5′-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

De novo RNA-Seq assembly facilitates the study of transcriptomes for species without sequenced genomes, but it is challenging to select the most accurate assembly in this context. To address this challenge, we developed a model-based score, RSEM-EVAL, for evaluating assemblies when the ground truth is unknown. We show that RSEM-EVAL correctly reflects assembly accuracy, as measured by REF-EVAL, a refined set of ground-truth-based scores that we also developed. Guided by RSEM-EVAL, we assembled the transcriptome of the regenerating axolotl limb; this assembly compares favorably to a previous assembly. A software package implementing our methods, DETONATE, is freely available at http://deweylab.biostat.wisc.edu/detonate.

Promoter architecture dictates cell-to-cell variability in gene expression.

Science. 2014 Dec 19;346(6216):1533-6

Authors: Jones DL, Brewster RC, Phillips R

Abstract
Variability in gene expression among genetically identical cells has emerged as a central preoccupation in the study of gene regulation; however, a divide exists between the predictions of molecular models of prokaryotic transcriptional regulation and genome-wide experimental studies suggesting that this variability is indifferent to the underlying regulatory architecture. We constructed a set of promoters in Escherichia coli in which promoter strength, transcription factor binding strength, and transcription factor copy numbers are systematically varied, and used messenger RNA (mRNA) fluorescence in situ hybridization to observe how these changes affected variability in gene expression. Our parameter-free models predicted the observed variability; hence, the molecular details of transcription dictate variability in mRNA expression, and transcriptional noise is specifically tunable and thus represents an evolutionarily accessible phenotypic parameter.

PMID: 25525251 [PubMed - in process]

Microexons Go Big.

Cell. 2014 Dec 18;159(7):1488-1489

Authors: Yang L, Chen L

Abstract
Microexons are frequently underestimated in transcriptome analyses. Two studies published in Cell and Genome Research now independently report the identification of hundreds of microexons. Alternative splicing of some microexons is regulated by neuronal-specific RNA-binding proteins and modifies the function of proteins involved in neurogenesis, with misregulation linked to autism.

PMID: 25525868 [PubMed - as supplied by publisher]

Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log₂ number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

Publication date: 18 December 2014
Source:Cell, Volume 159, Issue 7
Author(s): Manuel Irimia , Robert J. Weatheritt , Jonathan D. Ellis , Neelroop N. Parikshak , Thomas Gonatopoulos-Pournatzis , Mariana Babor , Mathieu Quesnel-Vallières , Javier Tapial , Bushra Raj , Dave O’Hanlon , Miriam Barrios-Rodiles , Michael J.E. Sternberg , Sabine P. Cordes , Frederick P. Roth , Jeffrey L. Wrana , Daniel H. Geschwind , Benjamin J. Blencowe
Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide “microexons” display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

Graphical abstract

Teaser

Hundreds of 3–27 nt neuronal-specific “microexons” in mammals are identified and characterized. They are frequently misregulated in autistic brains.