Predicting transfer RNA gene activity from sequence and genome context.

Transfer RNA (tRNA) genes are among the most highly transcribed genes in the genome owing to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with difficulty in measuring transcript abundance and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish sequence-based correlates to tRNA gene expression and develop a tRNA gene classification method that does not require, but benefits from, comparative genomic information and achieves accuracy comparable to molecular assays. We observe that guanine + cytosine (G + C) content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our tRNA gene activity predictions in conjunction with a comprehensive tRNA gene ortholog set spanning 29 placental mammals to estimate the evolutionary rate of functional changes among orthologs. Our method adds a new dimension to large-scale tRNA functional prediction and will help prioritize characterization of functional tRNA variants. Its simplicity and robustness should enable development of similar approaches for other clades, as well as exploration of functional diversification of members of large gene families.

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo.

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns). One of the earliest events in this process is recognition of the 3' splice site (3'ss) by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proofreading of 3'ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control and HNRNPA1 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3'ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3'ss.

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC.

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.

RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation.

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

The RNAissance family: SR proteins as multifaceted regulators of gene expression.

Serine and arginine-rich (SR) proteins play multiple roles in the eukaryotic gene expression pathway. Initially described as constitutive and alternative splicing factors, now it is clear that SR proteins are key determinants of exon identity and function as molecular adaptors, linking the pre-messenger RNA (pre-mRNA) to the splicing machinery. In addition, now SR proteins are implicated in many aspects of mRNA and noncoding RNA (ncRNA) processing well beyond splicing. These unexpected roles, including RNA transcription, export, translation, and decay, may prove to be the rule rather than the exception. To simply define, this family of RNA-binding proteins as splicing factors belies the broader roles of SR proteins in post-transcriptional gene expression.

Frac-seq reveals isoform-specific recruitment to polyribosomes.

Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5'UTR as well as Alu-elements and microRNA target sites in the 3'UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes.

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.