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Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral 1 – 5 . Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar—albeit nonidentical—between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral. A survey of 8,341 mutations in 21 yeast genes shows that synonymous mutations are nearly as harmful as nonsynonymous mutations, in part because they both affect the mRNA level of the gene mutated.

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates. Accurate regulation of protein concentrations according to changes in cell volume that accompany growth and changes in biosynthetic capacity is an important component of cellular homeostasis. Here, using the model organism S. cerevisiae , the authors show how histone production is quantitatively coupled to genome content through the intrinsic properties of histone promoters.

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA—including initiation, elongation, and termination—at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

Damage control: small RNAs involved in DNA repair in Neurospora RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to humans. High-throughput sequencing has highlighted a vast reservoir of small non-coding RNAs in animals and plants, many of whose function remains to be determined. Despite the conservation of the RNAi pathways, whether similar types of small RNAs exist in the lower eukaryotes has been largely unexplored. Now a novel class of small RNAs has been identified in the filamentous fungus Neurospora . Named qiRNAs, for their association with the Argonaute protein QDE-2, like QDE-2 they appear in response to DNA damage. At about 20 nucleotides long they are slightly shorter than Neurospora siRNAs. Neurospora RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs in DNA repair as inhibitors of protein translation. High-throughput sequencing has highlighted a vast reservoir of small non-coding RNAs, the function of which, for the most part, remains to be determined. Here a new class of small RNAs, termed qiRNAs, is identified from the fungus Neurospora . The production of qiRNAs is dependent on DNA damage, and it is proposed that they may have a role in the DNA damage response. RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing 1 , 2 , 3 , 4 . The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa . This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20–21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5′ end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.

Recent studies of transcription have revealed a level of complexity not previously appreciated even a few years ago, both in the intricate use of post-initiation control and the mass production of rapidly degraded transcripts. Dissection of these pathways requires strategies for precisely following transcripts as they are being produced. Here we present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that although promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus, nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo . A closer look at transcription Transcription, the creation of a complementary RNA copy of a sequence of a cell's DNA template, is emerging as a more complicated process than what was thought just a few years ago. For instance, RNA polymerase pausing is common, providing a focal point for various regulatory processes, and many transcripts are destined for rapid degradation. To study these phenomena, Stirling Churchman and Jonathan Weissman have developed a technique called native elongating transcript sequencing (NET-seq), capable of quantifying transcription with single nucleotide resolution. NET-seq is based on the sequencing of nascent transcripts associated with RNA polymerase II that are captured directly from live cells. They use the method to gain insights into polymerase pausing and backtracking, and the directionality of transcription in the yeast Saccharomyces cerevisiae . A novel technique called native elongating transcript sequencing (NET-seq) is described, which can quantify transcription with single nucleotide resolution. It is based on sequencing nascent transcripts associated with RNA polymerase II that are captured directly from live cells, and is used to gain insights into polymerase pausing and backtracking and the directionality of transcription.

To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress‐responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress‐induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA‐derived mRNA synthesis rates identified 16 stress‐specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems. Synopsis Nascent transcriptome analysis reveals dynamics of mRNA synthesis and decay in yeast. The first step in the expression of the genome is the synthesis of messenger‐RNA (mRNA). In all cells, the regulation of mRNA levels in response to changing environmental conditions is a fundamental process. Classical methods to study such changes in mRNA levels, however, fail to unravel whether such changes are due to changes in mRNA synthesis (transcription) or changes in mRNA decay, which both contribute to setting mRNA levels. Therefore, the regulation of mRNA stability and turnover is poorly understood, and new methods for a quantitative analysis of mRNA synthesis and decay are urgenlty sought. In this study, we describe a novel method termed dynamic transcriptome analysis (DTA), which can be used to determine synthesis and decay rates of mRNAs on a genome‐wide level in yeast and other eukaryotic cells. We applied DTA to the model organism Saccharomyces cerevisiae and analyzed the dynamics of the transcriptome under standard growth conditions as well as under osmotic stress conditions. DTA relies on a combination of biochemistry, high‐throughput data acquisition, and computational biology. It uses metabolic labeling of newly synthesised RNA with the nucleoside analogon 4‐thiouridine (4sU), purification of labeled, newly synthesized RNA, and subsequent microarray hybridization. An improved mathematical model enables synthesis and decay rates of esentially all mRNAs in the cell to be determined with accuracy. In this study, we found that under normal growth conditions the synthesis rates for most mRNAs are low and that the decay rates are not correlated with synthesis. Addition of salt to the culture, however, induced three phases of changes in mRNA synthesis and decay. During the initial shock phase, there is a global repression of synthesis and a reduction of decay of most mRNAs. The subsequent induction phase involves strongly increased synthesis of stress mRNAs, which are also destabilized. Finally, the recovery phase restores decay rates, but leaves synthesis rates altered, apparently to allow for cellular growth under the new conditions. DTA shows a higher sensitivity and better temporal resolution than classical methods such as transcriptomics. Also, DTA is non‐perturbing and allows for an unbiased monitoring of genomic regulatory systems in living cells. Previously used methods are invasive and likely alter cellular physiology and thereby mRNA dynamics. DTA has a high potential to become a standard technique in molecular biology that may replace standard transcriptomics to study gene regulatory systems. In the future, DTA may be used to study dynamic changes in cellular mRNA metabolism induced by chemical inhibitors or defined mutations or changes in the environment. Rates of mRNA synthesis and decay can be measured on a genome‐wide scale in yeast by dynamic transcriptome analysis (DTA), which combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA realistically monitors the cellular response to osmotic stress with higher sensitivity and temporal resolution than transcriptomics, and can be used to follow changes in RNA metabolism in gene regulatory systems.

During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes.

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

Genome replication introduces a stepwise increase in the DNA template available for transcription. Genes replicated early in S phase experience this increase before late-replicating genes, raising the question of how expression levels are affected by DNA replication. We show that in budding yeast, messenger RNA (mRNA) synthesis rate is buffered against changes in gene dosage during S phase. This expression homeostasis depends on acetylation of H3 on its internal K56 site by Rtt109/Asf1. Deleting these factors, mutating H3K56 or up-regulating its deacetylation, increases gene expression in S phase in proportion to gene replication timing. Therefore, H3K56 acetylation on newly deposited histones reduces transcription efficiency from replicated DNA, complementing its role in guarding genome stability. Our study provides molecular insight into the mechanism maintaining expression homeostasis during DNA replication.

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.