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.

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.

Location of polyadenylation sites within 3' untranslated regions is linked to biological function in yeast.

Expression of a typical yeast gene results in ∼50 3' mRNA isoforms that are distinguished by the locations of poly(A) sites within the 3' untranslated regions (3' UTRs). The location of poly(A) sites with respect to the translational termination codon varies considerably among genes, but whether this has any functional significance is poorly understood. Using hierarchical clustering of 3' UTRs, we identify eight classes of S. cerevisiae genes based on their poly(A) site locations. Genes involved in related biological functions (GO categories) are uniquely over-represented in six of these classes. Similar analysis of S. pombe genes reveals three classes of 3' UTRs, all of which show over-representation of functionally related genes. Remarkably, S. cerevisiae and S. pombe homologs share related patterns of poly(A) site locations. These observations suggest that the location of poly(A) sites within 3' UTRs has biological significance.

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.

Mot1 associates with transcriptionally active promoters and inhibits association of NC2 in Saccharomyces cerevisiae.

Mot1 stably associates with the TATA-binding protein (TBP), and it can dissociate TBP from DNA in an ATP-dependent manner. Mot1 acts as a negative regulator of TBP function in vitro, but genome-wide transcriptional profiling suggests that Mot1 positively affects about 10% of yeast genes and negatively affects about 5%. Unexpectedly, Mot1 associates with active RNA polymerase (Pol) II and III promoters, and it is rapidly recruited in response to activator proteins. At Pol II promoters, Mot1 association requires TBP and is strongly correlated with the level of TBP occupancy. However, the Mot1/TBP occupancy ratio at both Mot1-stimulated and Mot1-inhibited promoters is high relative to that at typical promoters, strongly suggesting that Mot1 directly affects transcriptional activity in a positive or negative manner, depending on the gene. The effect of Mot1 at the HIS3 promoter region depends on the functional quality and DNA sequence of the TATA element. Unlike TBP, Mot1 association is largely independent of the Srb4 component of Pol II holoenzyme, and it also can occur downstream of the promoter region. Mot1 removes TBP, but not TBP complexes or preinitiation complexes, from inappropriate genomic locations. Mot1 inhibits the association of NC2 with promoters, suggesting that the TBP-Mot1 and TBP-NC2 complexes compete for promoter occupancy in vivo. We speculate that Mot1 does not form transcriptionally active TBP complexes but rather regulates transcription in vivo by modulating the activity of free TBP and/or by affecting promoter DNA structure.

Quantitative sequential chromatin immunoprecipitation, a method for analyzing co-occupancy of proteins at genomic regions in vivo.

Sequential chromatin immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein-DNA complexes from living cells are subjected to two sequential immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.

Genome-Wide Study of mRNA Isoform Half-Lives.

In eukaryotes, RNA polymerase II-driven transcription and processing results in the formation of numerous mRNA 3' isoforms that for any given gene may differ from one another by as little as a single nucleotide. These 3' isoforms can vary in physical properties that may affect their function and stability. Here, we outline a systematic framework to measure individual mRNA 3' isoform half-lives on a genome-wide level in S. cerevisiae. Our approach utilizes the Anchor-Away system to sequester RNA polymerase II (Pol II) in the cytoplasm followed by direct single-molecule RNA sequencing to generate a highly detailed view of 3' isoform stability under most physiological conditions without many of the adverse effects associated with commonly used alternative approaches.

Mapping 3' mRNA isoforms on a genomic scale.

Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes. In recent years, a number of strategies utilizing high-throughput sequencing technologies have been developed to aid in the identification of genome-wide poly(A) sites. This unit describes a modified protocol for a recently published 3'READS (3' region extraction and deep sequencing) method that accurately identifies genome-wide poly(A) sites and that can be used to quantify the relative abundance of the resulting 3' mRNA isoforms. This approach minimizes nonspecific sequence reads due to internal priming and typically yields a high percentage of sequence reads that are ideally suited for accurate poly(A) identification.

Secondary structures involving the poly(A) tail and other 3' sequences are major determinants of mRNA isoform stability in yeast.

In Saccharomyces cerevisiae, previous measurements of mRNA stabilities have been determined on a per-gene basis. We and others have recently shown that yeast genes give rise to a highly heterogeneous population of mRNAs thanks to extensive alternative 3' end formation. Typical genes can have fifty or more distinct mRNA isoforms with 3' endpoints differing by as little as one and as many as hundreds of nucleotides. In our recent paper [Geisberg et al. Cell (2014) 156: 812-824] we measured half-lives of individual mRNA isoforms in Saccharomyces cerevisiae by using the anchor away method for the rapid removal of Rpb1, the largest subunit of RNA Polymerase II, from the nucleus, followed by direct RNA sequencing of the cellular mRNA population over time. Combining these two methods allowed us to determine half-lives for more than 20,000 individual mRNA isoforms originating from nearly 5000 yeast genes. We discovered that different 3' mRNA isoforms arising from the same gene can have widely different stabilities, and that such half-life variability across mRNA isoforms from a single gene is highly prevalent in yeast cells. Determining half-lives for many different mRNA isoforms from the same genes allowed us to identify hundreds of RNA sequence elements involved in the stabilization and destabilization of individual isoforms. In many cases, the poly(A) tail is likely to participate in the formation of stability-enhancing secondary structures at mRNA 3' ends. Our results point to an important role for mRNA structure at 3' termini in governing transcript stability, likely by reducing the interaction of the mRNA with the degradation apparatus.

Construction of mutant alleles in Saccharomyces cerevisiae without cloning: overview and the delitto perfetto method.

Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene.

Carryover test in frozen serum cored by an automated frozen sample aliquotter.

A cleaning protocol for the CryoXtract Automated Frozen Sample Aliquotter (AFSA) was tested for its effectiveness in eliminating sample-to-sample carryover during hands-free frozen sample aliquotting. The instrument's test platform was used to extract a statistically significant number of frozen cores from frozen bovine serum samples spiked with human DNA and from identical DNA-free frozen controls, processed in alternating succession, with a proprietary cleaning protocol performed in between samples. Measurements of DNA content by a highly sensitive real-time quantitative polymerase chain reaction assay revealed no traces of DNA in the frozen control cores, whereas they revealed the expected DNA amounts in all DNA-containing frozen cores. Analysis of high-copy DNA demonstrated that CryoXtract's AFSA's cleaning protocol effectively reduced DNA carryover by more than a million-fold.