Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here—each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.
Hyeshik Chang
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Single-cell RNA-seq: advances and future challenges
5' isomiR variation is of functional and evolutionary importance
We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5' differences and in support of this we report that a 5' isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5' isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.
Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay
A sensitive and highly multiplex method to directly measure RNA sequence abundance without requiring reverse transcription would be of value for a number of biomedical applications, including high throughput small molecule screening, pathogen transcript detection and quantification of short/degraded RNAs. RNA Annealing, Selection and Ligation (RASL) assays, which are based on RNA template-dependent oligonucleotide probe ligation, have been developed to meet this need, but technical limitations have impeded their adoption. Whereas DNA ligase-based RASL assays suffer from extremely low and sequence-dependent ligation efficiencies that compromise assay robustness, Rnl2 can join a fully DNA donor probe to a 3'-diribonucleotide-terminated acceptor probe with high efficiency on an RNA template strand. Rnl2-based RASL exhibits sub-femtomolar transcript detection sensitivity, and permits the rational tuning of probe signals for optimal analysis by massively parallel DNA sequencing (RASL-seq). A streamlined Rnl2-based RASL-seq protocol was assessed in a small molecule screen using 77 probe sets designed to monitor complex human B cell phenotypes during antibody class switch recombination. Our data demonstrate the robustness, cost-efficiency and broad applicability of Rnl2-based RASL assays.
The mRNP remodeling mediated by UPF1 promotes rapid degradation of replication-dependent histone mRNA
Histone biogenesis is tightly controlled at multiple steps to maintain the balance between the amounts of DNA and histone protein during the cell cycle. In particular, translation and degradation of replication-dependent histone mRNAs are coordinately regulated. However, the underlying molecular mechanisms remain elusive. Here, we investigate remodeling of stem-loop binding protein (SLBP)-containing histone mRNPs occurring during the switch from the actively translating mode to the degradation mode. The interaction between a CBP80/20-dependent translation initiation factor (CTIF) and SLBP, which is important for efficient histone mRNA translation, is disrupted upon the inhibition of DNA replication or at the end of S phase. This disruption is mediated by competition between CTIF and UPF1 for SLBP binding. Further characterizations reveal hyperphosphorylation of UPF1 by activated ATR and DNA-dependent protein kinase upon the inhibition of DNA replication interacts with SLBP more strongly, promoting the release of CTIF and eIF3 from SLBP-containing histone mRNP. In addition, hyperphosphorylated UPF1 recruits PNRC2 and SMG5, triggering decapping followed by 5'-to-3' degradation of histone mRNAs. The collective observations suggest that both inhibition of translation and recruitment of mRNA degradation machinery during histone mRNA degradation are tightly coupled and coordinately regulated by UPF1 phosphorylation.
Context-dependent control of alternative splicing by RNA-binding proteins
[Report] Genomic-scale exchange of mRNA between a parasitic plant and its hosts
[Special Issue Review] The biology of mammalian parenting and its effect on offspring social development
High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA-tRNA interface.
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High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA-tRNA interface.
Genome Res. 2014 Nov;24(11):1797-807
Authors: Schmitt BM, Rudolph KL, Karagianni P, Fonseca NA, White RJ, Talianidis I, Odom DT, Marioni JC, Kutter C
Abstract
The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we characterized the expression of mRNA and tRNA genes quantitatively at multiple time points in two developing mouse tissues. We discovered that mRNA codon pools are highly stable over development and simply reflect the genomic background; in contrast, precise regulation of tRNA gene families is required to create the corresponding tRNA transcriptomes. The dynamic regulation of tRNA genes during development is controlled in order to generate an anticodon pool that closely corresponds to messenger RNAs. Thus, across development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.
PMID: 25122613 [PubMed - indexed for MEDLINE]
Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.
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Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.
Cancer Cell. 2014 Aug 11;26(2):248-61
Authors: Nguyen LH, Robinton DA, Seligson MT, Wu L, Li L, Rakheja D, Comerford SA, Ramezani S, Sun X, Parikh MS, Yang EH, Powers JT, Shinoda G, Shah SP, Hammer RE, Daley GQ, Zhu H
Abstract
Lin28a/b are RNA-binding proteins that influence stem cell maintenance, metabolism, and oncogenesis. Poorly differentiated, aggressive cancers often overexpress Lin28, but its role in tumor initiation or maintenance has not been definitively addressed. We report that LIN28B overexpression is sufficient to initiate hepatoblastoma and hepatocellular carcinoma in murine models. We also detected Lin28b overexpression in MYC-driven hepatoblastomas, and liver-specific deletion of Lin28a/b reduced tumor burden, extended latency, and prolonged survival. Both intravenous siRNA against Lin28b and conditional Lin28b deletion reduced tumor burden and prolonged survival. Igf2bp proteins are upregulated, and Igf2bp3 is required in the context of LIN28B overexpression to promote growth. Therefore, multiple murine models demonstrate that Lin28b is both sufficient to initiate liver cancer and necessary for its maintenance.
PMID: 25117712 [PubMed - in process]
Stem-Loop Recognition by DDX17 Facilitates miRNA Processing and Antiviral Defense
Source:Cell, Volume 158, Issue 4
Author(s): Ryan H. Moy , Brian S. Cole , Ari Yasunaga , Beth Gold , Ganesh Shankarling , Andrew Varble , Jerome M. Molleston , Benjamin R. tenOever , Kristen W. Lynch , Sara Cherry
DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.
Graphical abstract
Teaser
DDX17, an evolutionarily conserved member of the DEAD-box family, has a dual role in the recognition of RNA stem loops. Whereas in the nucleus it binds the stem loops of host pri-miRNA and facilitates miRNA biogenesis, in the cytoplasm, it binds virally encoded stem loops to restrict infection in an interferon-independent manner.Considerations when investigating lncRNA function in vivo.
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Considerations when investigating lncRNA function in vivo.
Elife. 2014;3:e03058
Authors: Bassett AR, Akhtar A, Barlow DP, Bird AP, Brockdorff N, Duboule D, Ephrussi A, Ferguson-Smith AC, Gingeras TR, Haerty W, Higgs DR, Miska EA, Ponting CP
Abstract
Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.DOI: http://dx.doi.org/10.7554/eLife.03058.001.
PMID: 25124674 [PubMed - in process]
Transcriptional interference by antisense RNA is required for circadian clock function
Nature advance online publication 17 August 2014. doi:10.1038/nature13671
Authors: Zhihong Xue, Qiaohong Ye, Simon R. Anson, Jichen Yang, Guanghua Xiao, David Kowbel, N. Louise Glass, Susan K. Crosthwaite & Yi Liu
Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities. Natural antisense RNAs are found in a wide range of eukaryotic organisms. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear. frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop, which was thought to generate sustained rhythmicity. Transcription of qrf, the long non-coding frq antisense RNA, is induced by light, and its level oscillates in antiphase to frq sense RNA. Here we show that qrf transcription is regulated by both light-dependent and light-independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. Light-independent qrf expression, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modelling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription. Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.
Import routes and nuclear functions of Argonaute and other small RNA-silencing proteins
Source:Trends in Biochemical Sciences, Volume 39, Issue 9
Author(s): Daniel Schraivogel , Gunter Meister
Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus.
The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
Nature Medicine 20, 936 (2014). doi:10.1038/nm.3626
Authors: Jeongmin Ryoo, Jongsu Choi, Changhoon Oh, Sungchul Kim, Minji Seo, Seok-Young Kim, Daekwan Seo, Jongkyu Kim, Tommy E White, Alberto Brandariz-Nuñez, Felipe Diaz-Griffero, Cheol-Heui Yun, Joseph A Hollenbaugh, Baek Kim, Daehyun Baek & Kwangseog Ahn
The HIV-1 restriction factor SAM domain– and HD domain–containing protein 1 (SAMHD1) is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.
A new activity for SAMHD1 in HIV restriction
Nature Medicine 20, 808 (2014). doi:10.1038/nm.3657
Authors: Zhiyuan Yang & Warner C Greene
Restriction factors are host cell proteins that inhibit retroviral infection. A new study using mutants of human HIV-1 restriction factor SAMHD1 suggests that it inhibits infection through degradation of viral RNA rather than through its dNTPase activity as previously suggested.
snRNA quality control by decapping and 5'-3' decay [Biochemistry]
p53 is positively regulated by miR-542-3p.
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p53 is positively regulated by miR-542-3p.
Cancer Res. 2014 Jun 15;74(12):3218-27
Authors: Wang Y, Huang JW, Castella M, Huntsman DG, Taniguchi T
Abstract
The tumor suppressor p53 and miRNAs are linked through a complex network. Several miRNAs modulate p53 expression, while p53 regulates the transcription and/or biogenesis of several other miRNAs. Here, we report the development of a cell-based assay used with a library of human miRNA mimics in a high-throughput screen for miRNAs that modulate p53 expression. Overexpression of miRNA (miR)-542-3p in cancer cells elevated p53 expression, stimulated the expression of p53 targets, and inhibited cell proliferation. Mechanistically, miR-542-3p increased p53 protein stability by weakening interactions between p53 and its negative regulator MDM2. Furthermore, miR-542-3p suppressed ribosome biogenesis by downregulating a subset of ribosomal proteins such as RPS23, leading to upregulation of RPL11 and stabilization of p53. The 3'untranslated region in the RPS23 transcript contained a miR-542-3p-binding site, suggesting that RPS23 is a direct target of miR-542-3p. Our results define miR-542-3p as an important new positive regulator of p53 with potential applications in cancer treatment.
PMID: 24762395 [PubMed - in process]
Genome-wide profiling reveals a role for T-cell intracellular antigens TIA1 and TIAR in the control of translational specificity in HeLa cells.
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Genome-wide profiling reveals a role for T-cell intracellular antigens TIA1 and TIAR in the control of translational specificity in HeLa cells.
Biochem J. 2014 Jul 1;461(1):43-50
Authors: Carrascoso I, Sánchez-Jiménez C, Izquierdo JM
Abstract
TIA (T-cell intracellular antigens)-knockdown HeLa cells show an increase in ribosomes and translational machinery components. This increase correlates with specific changes in translationally up-regulated mRNAs involved in cell-cycle progression and DNA repair, as shown in polysomal profiling analysis. Our data support the hypothesis that a concerted activation of both global and selective translational rates leads to the transition to a more proliferative status in TIA-knockdown HeLa cells.
PMID: 24927121 [PubMed - in process]
Clonal evolution in breast cancer revealed by single nucleus genome sequencing
Clonal evolution in breast cancer revealed by single nucleus genome sequencing
Nature 512, 7513 (2014). doi:10.1038/nature13600
Authors: Yong Wang, Jill Waters, Marco L. Leung, Anna Unruh, Whijae Roh, Xiuqing Shi, Ken Chen, Paul Scheet, Selina Vattathil, Han Liang, Asha Multani, Hong Zhang, Rui Zhao, Franziska Michor, Funda Meric-Bernstam & Nicholas E. Navin
Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We
Chromatin enrichment for proteomics
Nature Protocols 9, 2090 (2014). doi:10.1038/nprot.2014.142
Authors: Georg Kustatscher, Karen L H Wills, Cristina Furlan & Juri Rappsilber
During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its
How chemistry supports cell biology: the chemical toolbox at your service
Source:Trends in Cell Biology, Volume 24, Issue 12
Author(s): Ruud H. Wijdeven , Jacques Neefjes , Huib Ovaa
Chemical biology is a young and rapidly developing scientific field. In this field, chemistry is inspired by biology to create various tools to monitor and modulate biochemical and cell biological processes. Chemical contributions such as small-molecule inhibitors and activity-based probes (ABPs) can provide new and unique insights into previously unexplored cellular processes. This review provides an overview of recent breakthroughs in chemical biology that are likely to have a significant impact on cell biology. We also discuss the application of several chemical tools in cell biology research.
Translation and silencing in RNA granules: a tale of sand grains.
Translation and silencing in RNA granules: a tale of sand grains.
Front Mol Neurosci. 2014;7:68
Authors: Pimentel J, Boccaccio GL
Abstract
The transcriptome at the synapse consists of thousands of messengers encoding several cellular functions, including a significant number of receptors and ion channels and associated proteins. The concerted translational regulation of all these molecules contributes to the dynamic control of synaptic strength. Cumulative evidence supports that dendritic RNA granules and mRNA-silencing foci play an important role in translational regulation. Several relevant RBPs - FMRP; FUS/TLS; TDP-43; Staufen; Smaug; Pumilio; CPEB; HuD; ZBP1; and DDX6 among others - form granules that contain dormant mRNAs repressed by multiple pathways. Recent reports indicate that dendritic granules may contain stalled polysomes, and furthermore, active translation may occur in association with RNA granules. Here, we discuss the molecules and pathways involved in this continuum of RNA granules that contain masked mRNAs, mRNAs trapped in inactive polysomes or mRNAs engaged in translation.
PMID: 25100944 [PubMed]
Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates
Nature advance online publication 10 August 2014. doi:10.1038/nature13590
Authors: Delphine Goubau, Martin Schlee, Safia Deddouche, Andrea J. Pruijssers, Thomas Zillinger, Marion Goldeck, Christine Schuberth, Annemarthe G. Van der Veen, Tsutomu Fujimura, Jan Rehwinkel, Jason A. Iskarpatyoti, Winfried Barchet, Janos Ludwig, Terence S. Dermody, Gunther Hartmann & Caetano Reis e Sousa
Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5′-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5′-diphosphates (5′pp). Genomes from mammalian reoviruses with 5′pp termini, 5′pp-RNA isolated from yeast L-A virus, and base-paired 5′pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5′pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5′pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5′pp-RNA, like that of 5′ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.
Influence of RNA extraction methods and library selection schemes on RNA-seq data
A protein-RNA specificity code enables targeted activation of an endogenous human transcript
Nature Structural & Molecular Biology 21, 732 (2014). doi:10.1038/nsmb.2847
Authors: Zachary T Campbell, Cary T Valley & Marvin Wickens
Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation
Nature Structural & Molecular Biology 21, 671 (2014). doi:10.1038/nsmb.2855
Authors: Andreas Schlundt, Gitta A Heinz, Robert Janowski, Arie Geerlof, Ralf Stehle, Vigo Heissmeyer, Dierk Niessing & Michael Sattler
Structural basis for activity of highly efficient RNA mimics of green fluorescent protein
Nature Structural & Molecular Biology 21, 658 (2014). doi:10.1038/nsmb.2865
Authors: Katherine Deigan Warner, Michael C Chen, Wenjiao Song, Rita L Strack, Andrea Thorn, Samie R Jaffrey & Adrian R Ferré-D'Amaré
Expanding the RNA-recognition code of PUF proteins
Nature Structural & Molecular Biology 21, 653 (2014). doi:10.1038/nsmb.2863
Author: Traci M Tanaka Hall
Classical PUF proteins bind to single-stranded RNA with sequence specificity that can be engineered by site-directed mutagenesis according to a simple RNA-recognition code. Now in-depth probing of the PUF RNA-recognition code enhances future design of PUF proteins and exposes hidden complexity in generating specificity.