Discovery of physiological and cancer-related regulators of 3' UTR processing with KAPAC.

3' Untranslated regions (3' UTRs) length is regulated in relation to cellular state. To uncover key regulators of poly(A) site use in specific conditions, we have developed PAQR, a method for quantifying poly(A) site use from RNA sequencing data and KAPAC, an approach that infers activities of oligomeric sequence motifs on poly(A) site choice. Application of PAQR and KAPAC to RNA sequencing data from normal and tumor tissue samples uncovers motifs that can explain changes in cleavage and polyadenylation in specific cancers. In particular, our analysis points to polypyrimidine tract binding protein 1 as a regulator of poly(A) site choice in glioblastoma.

A comprehensive analysis of 3' end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation.

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region (3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3' end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3' end processing in both mouse and human. With 3' end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3' UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3' UTR-dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3' end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3' end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle.

An updated human snoRNAome.

Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs that guide the post-transcriptional processing of other non-coding RNAs (mostly ribosomal RNAs), but have also been implicated in processes ranging from microRNA-dependent gene silencing to alternative splicing. In order to construct an up-to-date catalog of human snoRNAs we have combined data from various databases, de novo prediction and extensive literature review. In total, we list more than 750 curated genomic loci that give rise to snoRNA and snoRNA-like genes. Utilizing small RNA-seq data from the ENCODE project, our study characterizes the plasticity of snoRNA expression identifying both constitutively as well as cell type specific expressed snoRNAs. Especially, the comparison of malignant to non-malignant tissues and cell types shows a dramatic perturbation of the snoRNA expression profile. Finally, we developed a high-throughput variant of the reverse-transcriptase-based method for identifying 2'-O-methyl modifications in RNAs termed RimSeq. Using the data from this and other high-throughput protocols together with previously reported modification sites and state-of-the-art target prediction methods we re-estimate the snoRNA target RNA interaction network. Our current results assign a reliable modification site to 83% of the canonical snoRNAs, leaving only 76 snoRNA sequences as orphan.

Roquin recognizes a non-canonical hexaloop structure in the 3'-UTR of Ox40.

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3'-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.

Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data.

BACKGROUND: Understanding the regulation of gene expression, including transcription start site usage, alternative splicing, and polyadenylation, requires accurate quantification of expression levels down to the level of individual transcript isoforms. To comparatively evaluate the accuracy of the many methods that have been proposed for estimating transcript isoform abundance from RNA sequencing data, we have used both synthetic data as well as an independent experimental method for quantifying the abundance of transcript ends at the genome-wide level. RESULTS: We found that many tools have good accuracy and yield better estimates of gene-level expression compared to commonly used count-based approaches, but they vary widely in memory and runtime requirements. Nucleotide composition and intron/exon structure have comparatively little influence on the accuracy of expression estimates, which correlates most strongly with transcript/gene expression levels. To facilitate the reproduction and further extension of our study, we provide datasets, source code, and an online analysis tool on a companion website, where developers can upload expression estimates obtained with their own tool to compare them to those inferred by the methods assessed here. CONCLUSIONS: As many methods for quantifying isoform abundance with comparable accuracy are available, a user's choice will likely be determined by factors such as the memory and runtime requirements, as well as the availability of methods for downstream analyses. Sequencing-based methods to quantify the abundance of specific transcript regions could complement validation schemes based on synthetic data and quantitative PCR in future or ongoing assessments of RNA-seq analysis methods.

The ViennaRNA web services.

The ViennaRNA package is a widely used collection of programs for thermodynamic RNA secondary structure prediction. Over the years, many additional tools have been developed building on the core programs of the package to also address issues related to noncoding RNA detection, RNA folding kinetics, or efficient sequence design considering RNA-RNA hybridizations. The ViennaRNA web services provide easy and user-friendly web access to these tools. This chapter describes how to use this online platform to perform tasks such as prediction of minimum free energy structures, prediction of RNA-RNA hybrids, or noncoding RNA detection. The ViennaRNA web services can be used free of charge and can be accessed via http://rna.tbi.univie.ac.at.

Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells.

Alternative polyadenylation is a cellular mechanism that generates mRNA isoforms differing in their 3' untranslated regions (3' UTRs). Changes in polyadenylation site usage have been described upon induction of proliferation in resting cells, but the underlying mechanism and functional significance of this phenomenon remain largely unknown. To understand the functional consequences of shortened 3' UTR isoforms in a physiological setting, we used 3' end sequencing and quantitative mass spectrometry to determine polyadenylation site usage, mRNA and protein levels in murine and human naive and activated T cells. Although 3' UTR shortening in proliferating cells is conserved between human and mouse, orthologous genes do not exhibit similar expression of alternative 3' UTR isoforms. We generally find that 3' UTR shortening is not accompanied by a corresponding change in mRNA and protein levels. This suggests that although 3' UTR shortening may lead to changes in the RNA-binding protein interactome, it has limited effects on protein output.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.

RNA Polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes.

RNA Polymerase III is a highly specialized enzyme complex responsible for the transcription of a very distinct set of housekeeping noncoding RNAs including tRNAs, 7SK snRNA, Y RNAs, U6 snRNA, and the RNA components of RNaseP and RNaseMRP. In this work we have utilized the conserved promoter structure of known RNA Polymerase III transcripts consisting of characteristic sequence elements termed proximal sequence elements (PSE) A and B and a TATA-box to uncover a novel RNA Polymerase III-transcribed, noncoding RNA family found to be conserved in Caenorhabditis as well as other clade V nematode species. Homology search in combination with detailed sequence and secondary structure analysis revealed that members of this novel ncRNA family evolve rapidly, and only maintain a potentially functional small stem structure that links the 5' end to the very 3' end of the transcript and a small hairpin structure at the 3' end. This is most likely required for efficient transcription termination. In addition, our study revealed evidence that canonical C/D box snoRNAs are also transcribed from a PSE A-PSE B-TATA-box promoter in Caenorhabditis elegans.

Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors.

Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and polyadenylation. A core 3'-end processing complex carries out the cleavage and polyadenylation reactions, but many proteins have been implicated in the selection of polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3'-end processing sites as well as the binding sites of proteins that are involved in the selection of cleavage sites and in the processing reactions have been developed. Here, we review these approaches as well as the insights into the mechanisms of polyadenylation that emerged from genome-wide studies of polyadenylation across a range of cell types and states.

Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs.

Recruitment of the UPF1 nonsense-mediated mRNA decay (NMD) factor to target mRNAs was initially proposed to occur through interaction with release factors at terminating ribosomes. However, recently emerging evidence points toward translation-independent interaction with the 3' untranslated region (UTR) of mRNAs. We mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation in human cells and found that UPF1 preferentially associated with 3' UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition, thus indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination.

Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing.

BACKGROUND: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. RESULTS: Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. CONCLUSIONS: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.

Cleavage factor Im is a key regulator of 3' UTR length.

In eukaryotes, the 3' ends of RNA polymerase II-transcribed RNAs are generated in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. Through alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control and drastic changes in the length of mRNA 3' UTRs upon induction of proliferation in resting cells. To understand the dynamics of poly(A) site choice, we recently identified binding sites of the major pre-mRNA 3' end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3' UTR length. More specifically, we demonstrate that the loss-of-function of CF Im 68 and CF Im 25 but not of CF Im 59 leads to a transcriptome-wide increase in the use of proximal polyadenylation sites in HEK293 cells.

Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length.

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF I(m)25, CF I(m)59, and CF I(m)68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFI(m) proteins have much higher positional specificity compared to CPSF components. Knockdown of CF I(m)68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF I(m)68 knockdown.

Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation.

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.

Animal snoRNAs and scaRNAs with exceptional structures.

The overwhelming majority of small nucleolar RNAs (snoRNAs) fall into two clearly defined classes characterized by distinctive secondary structures and sequence motifs. A small group of diverse ncRNAs, however, shares the hallmarks of one or both classes of snoRNAs but differs substantially from the norm in some respects. Here, we compile the available information on these exceptional cases, conduct a thorough homology search throughout the available metazoan genomes, provide improved and expanded alignments, and investigate the evolutionary histories of these ncRNA families as well as their mutual relationships.

AREsite: a database for the comprehensive investigation of AU-rich elements.

AREsite is an online resource for the detailed investigation of AU-rich elements (ARE) in vertebrate mRNA 3'-untranslated regions (UTRs). AREs are one of the most prominent cis-acting regulatory elements found in 3'-UTRs of mRNAs. Various ARE-binding proteins that possess RNA stabilizing or destabilizing functions are recruited by sequence-specific motifs. Recent findings suggest an essential role of the structural mRNA context in which these sequence motifs are embedded. AREsite is the first database that allows to quantify the structuredness of ARE motif sites in terms of opening energies and accessibility probabilities. Moreover, we also provide a detailed phylogenetic analysis of ARE motifs and incorporate information about experimentally validated targets of the ARE-binding proteins TTP, HuR and Auf1. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.

Nematode sbRNAs: homologs of vertebrate Y RNAs.

Stem-bulge RNAs (sbRNAs) are a group of small, functionally yet uncharacterized noncoding RNAs first described in C. elegans, with a few homologous sequences postulated in C. briggsae. In this study, we report on a comprehensive survey of this ncRNA family in the phylum Nematoda. Employing homology search strategies based on both sequence and secondary structure models and a computational promoter screen we identified a total of 240 new sbRNA homologs. For the majority of these loci we identified both promoter regions and transcription termination signals characteristic for pol-III transcripts. Sequence and structure comparison with known RNA families revealed that sbRNAs are homologs of vertebrate Y RNAs. Most of the sbRNAs show the characteristic Ro protein binding motif, and contain a region highly similar to a functionally required motif for DNA replication previously thought to be unique to vertebrate Y RNAs. The single Y RNA that was previously described in C. elegans, however, does not show this motif, and in general bears the hallmarks of a highly derived family member.

RNAz 2.0: improved noncoding RNA detection.

RNAz is a widely used software package for de novo detection of structured noncoding RNAs in comparative genomics data. Four years of experience have not only demonstrated the applicability of the approach, but also helped us to identify limitations of the current implementation. RNAz 2.0 provides significant improvements in two respects: (1) The accuracy is increased by the systematic use of dinucleotide models. (2) Technical limitations of the previous version, such as the inability to handle alignments with more than six sequences, are overcome by increased training data and the usage of an entropy measure to represent sequence similarities. RNAz 2.0 shows a significantly lower false discovery rate on a dinucleotide background model than the previous version. Separate models for structural alignments provide an additional way to increase the predictive power. RNAz is open source software and can be obtained free of charge at: http://www.tbi.univie.ac.at/~wash/RNAz/

RNAalifold: improved consensus structure prediction for RNA alignments.

BACKGROUND: The prediction of a consensus structure for a set of related RNAs is an important first step for subsequent analyses. RNAalifold, which computes the minimum energy structure that is simultaneously formed by a set of aligned sequences, is one of the oldest and most widely used tools for this task. In recent years, several alternative approaches have been advocated, pointing to several shortcomings of the original RNAalifold approach. RESULTS: We show that the accuracy of RNAalifold predictions can be improved substantially by introducing a different, more rational handling of alignment gaps, and by replacing the rather simplistic model of covariance scoring with more sophisticated RIBOSUM-like scoring matrices. These improvements are achieved without compromising the computational efficiency of the algorithm. We show here that the new version of RNAalifold not only outperforms the old one, but also several other tools recently developed, on different datasets. CONCLUSION: The new version of RNAalifold not only can replace the old one for almost any application but it is also competitive with other approaches including those based on SCFGs, maximum expected accuracy, or hierarchical nearest neighbor classifiers.