Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast.

We measured half-lives of 21,248 mRNA 3' isoforms in yeast by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process. Interestingly, half-lives of mRNA isoforms from the same gene, including nearly identical isoforms, often vary widely. Based on clusters of isoforms with different half-lives, we identify hundreds of sequences conferring stabilization or destabilization upon mRNAs terminating downstream. One class of stabilizing element is a polyU sequence that can interact with poly(A) tails, inhibit the association of poly(A)-binding protein, and confer increased stability upon introduction into ectopic transcripts. More generally, destabilizing and stabilizing elements are linked to the propensity of the poly(A) tail to engage in double-stranded structures. Isoforms engineered to fold into 3' stem-loop structures not involving the poly(A) tail exhibit even longer half-lives. We suggest that double-stranded structures at 3' ends are a major determinant of mRNA stability.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

Extensive Structural Differences of Closely Related 3' mRNA Isoforms: Links to Pab1 Binding and mRNA Stability.

Alternative polyadenylation generates numerous 3' mRNA isoforms that can vary in biological properties, such as stability and localization. We developed methods to obtain transcriptome-scale structural information and protein binding on individual 3' mRNA isoforms in vivo. Strikingly, near-identical mRNA isoforms can possess dramatically different structures throughout the 3' UTR. Analyses of identical mRNAs in different species or refolded in vitro indicate that structural differences in vivo are often due to trans-acting factors. The level of Pab1 binding to poly(A)-containing isoforms is surprisingly variable, and differences in Pab1 binding correlate with the extent of structural variation for closely spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-strandedness of the poly(A) tail, and low Pab1 binding is associated with mRNA stability. Thus, individual 3' mRNA isoforms can be remarkably different physical entities in vivo. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, indicating biological function.

Condition-specific 3' mRNA isoform half-lives and stability elements in yeast.

Alternative polyadenylation generates numerous 3' mRNA isoforms that can differ in their stability, structure, and function. These isoforms can be used to map mRNA stabilizing and destabilizing elements within 3' untranslated regions (3'UTRs). Here, we examine how environmental conditions affect 3' mRNA isoform turnover and structure in yeast cells on a transcriptome scale. Isoform stability broadly increases when cells grow more slowly, with relative half-lives of most isoforms being well correlated across multiple conditions. Surprisingly, dimethyl sulfate probing reveals that individual 3' isoforms have similar structures across different conditions, in contrast to the extensive structural differences that can exist between closely related isoforms in an individual condition. Unexpectedly, most mRNA stabilizing and destabilizing elements function only in a single growth condition. The genes associated with some classes of condition-specific stability elements are enriched for different functional categories, suggesting that regulated mRNA stability might contribute to adaptation to different growth environments. Condition-specific stability elements do not result in corresponding condition-specific changes in steady-state mRNA isoform levels. This observation is consistent with a compensatory mechanism between polyadenylation and stability, and it suggests that condition-specific mRNA stability elements might largely reflect condition-specific regulation of mRNA 3' end formation.

A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast.

Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and messenger RNA (mRNA) turnover in the cytoplasm. On a global basis, same-gene 3' mRNA isoforms with twofold or greater differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of 2. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3' mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability.

Elongation rate of RNA polymerase II affects pausing patterns across 3' UTRs.

Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.

Genome-wide oscillations in G + C density and sequence conservation.

Eukaryotic genomes typically show a uniform G + C content among chromosomes, but on smaller scales, many species have a G + C density that fluctuates with a characteristic wavelength. This oscillation is evident in many insect species, with wavelengths ranging between 700 bp and 4 kb. Measures of evolutionary conservation oscillate in phase with G + C content, with conserved regions having higher G + C. Loci with large regulatory regions show more regular oscillations; coding sequences and heterochromatic regions show little or no oscillation. There is little oscillation in vertebrate genomes in regions with densely distributed mobile repetitive elements. However, species with few repeats show oscillation in both G + C density and sequence conservation. These oscillations may reflect optimal spacing of cis-regulatory elements.

SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

Species-specific factors mediate extensive heterogeneity of mRNA 3' ends in yeasts.

Most eukaryotic genes express mRNAs with alternative polyadenylation sites at their 3' ends. Here we show that polyadenylated 3' termini in three yeast species (Saccharomyces cerevisiae, Kluyveromyces lactis, and Debaryomyces hansenii) are remarkably heterogeneous. Instead of a few discrete 3' ends, the average yeast gene has an "end zone," a >200 bp window with >60 distinct poly(A) sites, the most used of which represents only 20% of the mRNA molecules. The pattern of polyadenylation within this zone varies across species, with D. hansenii possessing a higher focus on a single dominant point closer to the ORF terminus. Some polyadenylation occurs within mRNA coding regions with a strong bias toward the promoter. The polyadenylation pattern is determined by a highly degenerate sequence over a broad region and by a local sequence that relies on A residues after the cleavage point. Many dominant poly(A) sites are predicted to adopt a common secondary structure that may be recognized by the cleavage/polyadenylation machinery. We suggest that the end zone reflects a region permissive for polyadenylation, within which cleavage occurs preferentially at the A-rich sequence. In S. cerevisiae strains, D. hansenii genes adopt the S. cerevisiae polyadenylation profile, indicating that the polyadenylation pattern is mediated primarily by species-specific factors.

High-throughput sequencing reveals a simple model of nucleosome energetics.

We use genome-wide nucleosome maps to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a minor role. Furthermore, similar sequence signatures are exhibited by control experiments in which nucleosome-free genomic DNA is either sonicated or digested with micrococcal nuclease, making it possible that current predictions based on high-throughput nucleosome-positioning maps are biased by experimental artifacts.

Close association of RNA polymerase II and many transcription factors with Pol III genes.

Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using alpha-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression.

Nucleosome depletion at yeast terminators is not intrinsic and can occur by a transcriptional mechanism linked to 3'-end formation.

Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone-DNA interactions. Rapid removal of RNA polymerase II (pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of pol II, but rather involves a distinct mechanism linked to 3'-end formation.

Mapping accessible chromatin regions using Sono-Seq.

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a size-selection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP-Seq experiments and the value of reference samples that should be used in such experiments.

3' Untranslated Regions Are Modular Entities That Determine Polyadenylation Profiles.

The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated regions (3' UTRs) but rarely within coding regions. An individual gene can yield many 3'-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3' UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3' UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3'-UTR chimeric genes indicate that the poly(A) profile is determined by the 3' UTR. Thus, 3' UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3'-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3' UTRs.

Nucleotide-level linkage of transcriptional elongation and polyadenylation.

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

The transcriptional elongation rate regulates alternative polyadenylation in yeast.

Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3' mRNA isoforms for most genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.

Protein Binding to mRNA 3' Isoforms.

Here we describe CLIP-READS, a technique that combines elements of crosslinking and immunoprecipitation (CLIP) and 3' region extraction and deep sequencing (READS), to provide a genome-wide map of mRNA 3' isoform binding by a given messenger ribonucleoprotein (mRNP). In CLIP-READS, cells are grown to logarithmic phase and are irradiated with UV light (254 nm) to form RNA-protein adducts. The protein-mRNA complexes are immunoprecipitated from cell extracts with an antibody specific to the protein of interest, after which the protein component is digested away with Pronase. Messenger RNAs are then subjected to 3' READS. An input sample processed by 3' READS in parallel allows for the relative quantification of isoform-specific binding by the mRNP of interest. © 2019 by John Wiley & Sons, Inc.

Probing In Vivo Structure of Individual mRNA 3' Isoforms Using Dimethyl Sulfate.

The DMS region extraction and deep sequencing (DREADS) procedure was designed to probe RNA structure in vivo and to link this structural information to specific 3' isoforms. Growing cells are treated with the alkylating agent dimethyl sulfate (DMS), which enters easily into cells and modifies RNA molecules at solvent-exposed A and C residues. RNA is isolated, and sequencing libraries are constructed in a manner that preserves the identities of individual mRNA isoforms arising from alternative cleavage/polyadenylation sites. During the cDNA synthesis step of library construction, the progress of reverse transcriptase (RT) is blocked when it encounters a DMS modification on the RNA, leading to disproportionate cDNA termination adjacent to DMS-modified positions. After paired-end deep sequencing, the downstream end of each sequenced fragment is mapped to a specific cleavage/poly(A) site representing an individual mRNA 3' isoform. The upstream mapped end of the sequenced fragment defines where the RT reaction stopped. Over the population of all sequenced fragments derived from a particular isoform, A and C positions that are overrepresented next to the upstream endpoints in the DMS sample (relative to a parallel untreated control) are inferred to have been DMS modified, and hence solvent exposed. This method thus allows in vivo structural information obtained using DMS to be linked to individual mRNA 3' isoforms. © 2019 by John Wiley & Sons, Inc.

Expanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast.

Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins.

Genome-wide occupancy profile of the RNA polymerase III machinery in Saccharomyces cerevisiae reveals loci with incomplete transcription complexes.

We used chromatin immunoprecipitation, followed by microarray hybridization, to determine the genome-wide distribution of the RNA polymerase (Pol) III transcription apparatus in the yeast Saccharomyces cerevisiae. The Pol III transcriptome includes all tRNA genes, previously identified non-tRNA Pol III genes, and SNR52, which encodes a small nucleolar RNA. Unexpectedly, we identify eight ETC loci that are occupied by TFIIIC but not by other components of the Pol III machinery. Some ETC loci contain stretches of DNA that are highly conserved among closely related yeast species, suggesting that they may encode functional RNAs. ETC6 is located upstream of the gene encoding the tau 91 subunit of TFIIIC, suggesting the possibility of Pol III-regulated expression of a critical Pol III factor. We also identify the ZOD1 locus, which is bound by all components of the Pol III machinery and yet does not appear to express an RNA conserved among closely related yeast species. The B block motifs and several flanking nucleotides of the ZOD1 and ETC loci are very similar to each other and are highly conserved across the yeast species. Furthermore, the unusual profile of Pol III factor association with ZOD1 and the ETC loci is perfectly preserved in a different Saccharomyces species, indicating that these loci represent novel functional entities.