4-Thiouridine Labeling to Analyze mRNA Turnover in Schizosaccharomyces pombe.

Traditionally, the half-lives of mRNAs were measured after inhibition of transcription to allow decay of the preexisting population. The protocol presented here is a more recently developed strategy in which mRNA turnover is analyzed by measuring the decline in levels of newly synthesized RNA labeled with 4-thiouridine (4sU) during a brief pulse. After RNA extraction, the 4sU is biotinylated and the labeled species are purified using streptavidin beads. DNA microarrays can then be used to compare this population with total RNA, allowing half-lives to be calculated.

Analysis of RNA Metabolism in Fission Yeast.

Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles.

Preparation of Total RNA from Fission Yeast.

Treatment with hot phenol breaks open fission yeast cells and begins to strip away bound proteins from RNA. Deproteinization is completed by multiple extractions with chloroform/isoamyl alcohol and separation of the aqueous and organic phases using MaXtract gel, an inert material that acts as a physical barrier between the phases. The final step is concentration of the RNA by ethanol precipitation. The protocol can be used to prepare RNA from several cultures grown in parallel, but it is important not to process too many samples at once because delays can be detrimental to RNA quality. A reasonable number of samples to process at once would be three to four for microarray or RNA sequencing analyses and six for preliminary investigations of mutants implicated in RNA metabolism.

Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis.

Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms.

The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin.

Measurement of in vivo RNA synthesis rates.

A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006).

A dominant role for meiosis-specific 3' RNA processing in controlling expression of a fission yeast cyclin gene.

Meiotic gene regulation provides a rich source of insight into mechanisms of temporal control during development. We previously reported that accumulation of many meiotic mRNAs in fission yeast is governed by changes in 3' RNA processing and elucidated the molecular basis of this regulatory mechanism for an early meiotic gene. Here, we report that cleavage/polyadenylation is also the nexus of negative control for middle meiotic genes. Parallel profiles of splicing and polyadenylation are observed over a meiotic time course for both rem1 and spo4 but not for a constitutive control gene. Nevertheless, polyadenylation of rem1 transcripts is restricted to meiosis by a splicing-independent mechanism. Through systematic sequence substitutions, we identified a negative control region (NCR) located upstream of the rem1 transcription start site and found that it is required to block 3' RNA processing in proliferating cells. Ablation of the NCR relieves inhibition regardless of whether the intron is present, absent, or carries splice site mutations. Consistent with the previous report of a polypeptide encoded by the first exon of rem1, we discovered a second 3' processing site just downstream from the 5' splice site. Polyadenylation within the intron is activated concurrent with the downstream site during meiosis, is controlled by the NCR, and is enhanced when splicing is blocked via 5' junction or branch point mutations. Taken together, these data suggest a novel regulatory mechanism in which a 5' element modulates the dynamic interplay between splicing and polyadenylation.

A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts.

To determine the relative importance of transcriptional regulation versus RNA processing and turnover during the transition from proliferation to meiotic differentiation in the fission yeast Schizosaccharomyces pombe, we analyzed temporal profiles and effects of RNA surveillance factor mutants on expression of 32 meiotic genes. A comparison of nascent transcription with steady-state RNA accumulation reveals that the vast majority of these genes show a lag between maximal RNA synthesis and peak RNA accumulation. During meiosis, total RNA levels parallel 3' processing, which occurs in multiple, temporally distinct waves that peak from 3 to 6 h after meiotic induction. Most early genes and one middle gene, mei4, share a regulatory mechanism in which a specialized RNA surveillance factor targets newly synthesized transcripts for destruction. Mei4p, a member of the forkhead transcription factor family, in turn regulates a host of downstream genes. Remarkably, a spike in transcription is observed for less than one-third of the genes surveyed, and even these show evidence of RNA-level regulation. In aggregate, our findings lead us to propose that a regulatory cascade driven by changes in processing and stability of newly synthesized transcripts operates alongside the well-known transcriptional cascade as fission yeast cells enter meiosis.

A complex gene regulatory mechanism that operates at the nexus of multiple RNA processing decisions.

Expression of crs1 pre-mRNA, encoding a meiotic cyclin, is blocked in actively growing fission yeast cells by a multifaceted mechanism. The most striking feature is that in vegetative cells, crs1 transcripts are continuously synthesized but are targeted for degradation rather than splicing and polyadenylation. Turnover of crs1 RNA requires the exosome, as do previously described nuclear surveillance and silencing mechanisms, but does not involve a noncanonical poly(A) polymerase. Instead, crs1 transcripts are targeted for destruction by a factor previously implicated in turnover of meiotic RNAs in growing cells. Like exosome mutants, mmi1 mutants splice and polyadenylate vegetative crs1 transcripts. Two regulatory elements are located at the 3' end of the crs1 gene, consistent with the increased accumulation of spliced RNA in polyadenylation factor mutants. This highly integrated regulatory strategy may ensure a rapid response to adverse conditions, thereby guaranteeing survival.

Negative control contributes to an extensive program of meiotic splicing in fission yeast.

Despite a high frequency of introns in the fission yeast Schizosaccharomyces pombe, regulated splicing is virtually unknown. We present evidence that splicing constitutes a major mechanism for controlling gene expression during meiosis, as 12 of 96 transcripts tested, which encode known components as well as previously uncharacterized ORFs, retain introns until specific times during differentiation. The meiotically spliced pre-mRNAs include two cyclins, rem1 (discovered by Ayte and Nurse) and crs1. Consistent with the use of regulated splicing to block protein production, expression of crs1 in vegetative cells is toxic. Analyses of gene chimeras indicate that splicing is prevented in mitotically growing cells via inhibition, in contrast to the positive control of meiotic splicing in budding yeast. Most strikingly, splicing of crs1 and rem1 is regulated by sequences located outside the coding regions, far from the target introns, a phenomenon previously observed only in metazoans.

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin.

Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3'-splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.

Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo.

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans.

The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF(SM)) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AF(SM) depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.