Aino I Järvelin

Polyadenylation site–induced decay of upstream transcripts enforces promoter directionality

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3′ ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Functional consequences of bidirectional promoters.

Several studies have shown that promoters of protein-coding genes are origins of pervasive non-coding RNA transcription and can initiate transcription in both directions. However, only recently have researchers begun to elucidate the functional implications of this bidirectionality and non-coding RNA production. Increasing evidence indicates that non-coding transcription at promoters influences the expression of protein-coding genes, revealing a new layer of transcriptional regulation. This regulation acts at multiple levels, from modifying local chromatin to enabling regional signal spreading and more distal regulation. Moreover, the bidirectional activity of a promoter is regulated at multiple points during transcription, giving rise to diverse types of transcripts.

An efficient method for genome-wide polyadenylation site mapping and RNA quantification

The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3′T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3′-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3′-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.

Alternative polyadenylation diversifies post‐transcriptional regulation by selective RNA–protein interactions

Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non‐coding transcriptional events from each genic locus using a novel genome‐wide, nucleotide‐resolution technique to quantify the half‐lives of 3′ transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3′ untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3′ UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA‐protein interactions in conditioning isoform‐specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

The new (dis)order in RNA regulation.

RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox interactions will yield important insights into RNA regulation in cellular homeostasis and disease.

Single-cell polyadenylation site mapping reveals 3' isoform choice variability.

Cell‐to‐cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3′ untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3′ isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome‐wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single‐cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single‐cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3′ isoform choice has potential implications on functional cell‐to‐cell heterogeneity as well as utility in resolving cell populations.

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap

This protocol is an extension to: Nat. Protoc. 8, 491–500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein–RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein–RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer

Cancer development involves the stepwise accumulation of genetic lesions that overcome the normal regulatory pathways that prevent unconstrained cell division and tissue growth. Identification of the genetic changes that cause cancer has long been the subject of intensive study, leading to the identification of several RNA-binding proteins (RBPs) linked to cancer. Cross-reference of the complement of RBPs recently identified by RNA interactome capture with cancer-associated genes and biological processes led to the identification of a set of 411 proteins with potential implications in cancer biology. These involve a broad spectrum of cellular processes including response to stress, metabolism and cell adhesion. Future studies should aim to understand these proteins and their connection to cancer from an RNA-centred perspective, holding the promise of new mechanistic understanding of cancer formation and novel approaches to diagnosis and treatment.

Regulating prospero mRNA Stability Determines When Neural Stem Cells Stop Dividing

During Drosophila and vertebrate brain development, termination of neural stem cell (neuroblast) proliferation and their differentiation require the conserved transcription factor Prospero/Prox1. It is not known how the level of Prospero is regulated to produce an expression peak in pupal neuroblasts, which terminates proliferation. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons and terminal pupal neuroblasts selectively transcribe a long prospero isoform containing a 15 kb 3′ UTR stabilised by binding to the conserved RNA-binding protein Syncrip/hnRNPQ. The long prospero isoform and Syncrip are both required to stop neuroblasts dividing. Our results demonstrate an unexpected function for mRNA stability in limiting neuroblast proliferation required for normal brain development. Given that Prox1 regulates vertebrate neuroblasts and other stem cells, our findings suggest widespread roles for regulated mRNA stability in stem cell biology.During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3’ untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prospero isoform, which allows an upregulation of Prospero protein production. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.