Mordechai Choder

Cytoplasmic 5 -3 exonuclease Xrn1p is also a genome-wide transcription factor in yeast

The 5′ to 3′ exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5′ region of a number of genes. In the present work, we show that disruption of Xrn1 activity preferentially affects both the synthesis and decay of a distinct subpopulation of mRNAs. The most affected mRNAs are the transcripts of the highly transcribed genes, mainly those encoding ribosome biogenesis and translation factors. Previously, we proposed that synthegradases play a key role in regulating both mRNA synthesis and degradation. Evidently, Xrn1 functions as a synthegradase, whose selectivity might help coordinating the expression of the protein synthetic machinery. We propose to name the most affected genes “Xrn1 synthegradon.”

The Fission Yeast TOR Homolog,tor1 +, Is Required for the Response to Starvation and Other Stresses via a Conserved Serine

Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth. Here we report the initial characterization of twoSchizosaccharomyces pombe TOR homologs,tor1 + and tor2 +.tor2 + is an essential gene, whereastor1 + is required only under starvation and other stress conditions. Specifically, Δtor1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress. In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs. Mutations at a conserved serine within the FRB domain ofSaccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins. The S. pombe tor1 + exhibits different features; substitution of the conserved serine residue, Ser1834, with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S. pombe.

SERKAL Syndrome: An Autosomal-Recessive Disorder Caused by a Loss-of-Function Mutation in WNT4

The WNT-signaling pathway plays a major role during mammalian embryogenesis. We report a novel autosomal-recessive syndrome that consists of female to male sex reversal and renal, adrenal, and lung dysgenesis and is associated with additional developmental defects. Using a candidate-gene approach, we identified a disease-causing homozygous missense mutation in the human WNT4 gene. The mutation was found to result in markedly reduced WNT4 mRNA levels in vivo and in vitro and to downregulate WNT4-dependent inhibition of beta-catenin degradation. Taken together with previous observations in animal models, the present data attribute a pivotal role to WNT4 signaling during organogenesis in humans.

Promoter Elements Regulate Cytoplasmic mRNA Decay

Promoters are DNA elements that enable transcription and its regulation by trans-acting factors. Here, we demonstrate that yeast promoters can also regulate mRNA decay after the mRNA leaves the nucleus. A conventional yeast promoter consists of a core element and an upstream activating sequence (UAS). We find that changing UASs of a reporter gene without altering the transcript sequence affects the transcripts decay kinetics. A short cis element, comprising two Rap1p-binding sites, and Rap1p itself, are necessary and sufficient to induce enhanced decay of the reporter mRNA. Furthermore, Rap1p stimulates both the synthesis and the decay of a specific population of endogenous mRNAs. We propose that Rap1p association with target promoter in the nucleus affects the composition of the exported mRNP, which in turn regulates mRNA decay in the cytoplasm. Thus, promoters can play key roles in determining mRNA levels and have the capacity to coordinate rates of mRNA synthesis and decay.

A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival.

Cells respond to stress by altering gene expression, and these adjustments facilitate stress tolerance. Although transcriptional changes are integral to most stress responses, little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Here we report that a major role of the RNA polymerase II subunit RPB4 is to permit appropriate transcriptional responses during stress. Yeast cells lacking RPB4 have essentially wild-type growth rates at moderate temperatures (18 to 22 degrees C), but their growth rates are substantially reduced at temperatures outside this range. When subjected to a heat shock, cells lacking RPB4 rapidly lose the ability to transcribe genes and experience a dramatic loss in viability. When cells lacking RPB4 are subjected to the nutrient stress that accompanies entry into stationary phase, they also exhibit a substantial decline in mRNA synthesis and in viability relative to wild-type cells. Interestingly, the portion of RNA polymerase II molecules that contain RPB4 is small in log phase but increases substantially as cells enter stationary phase. We propose that the association of RPB4 with the other RNA polymerase II subunits increases the tolerance of the enzyme to stress.

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis.

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.

Transcription in the nucleus and mRNA decay in the cytoplasm are coupled processes

Maintaining appropriate mRNAs levels is vital for any living cell. mRNA synthesis in the nucleus by RNA polymerase II core enzyme (Pol II) and mRNA decay by cytoplasmic machineries determine these levels. Yet, little is known about possible cross-talk between these processes. The yeast Rpb4/7 is a nucleo-cytoplasmic shuttling heterodimer that interacts with Pol II and with mRNAs and is required for mRNA decay in the cytoplasm. Here we show that interaction of Rpb4/7 with mRNAs and eventual decay of these mRNAs in the cytoplasm depends on association of Rpb4/7 with Pol II in the nucleus. We propose that, following its interaction with Pol II, Rpb4/7 functions in transcription, interacts with the transcript cotranscriptionally and travels with it to the cytoplasm to stimulate mRNA decay. Hence, by recruiting Rpb4/7, Pol II governs not only transcription but also mRNA decay.

Eukaryotic mRNA Decay: Methodologies, Pathways, and Links to Other Stages of Gene Expression

mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.

Transcriptome Kinetics Is Governed by a Genome-Wide Coupling of mRNA Production and Degradation: A Role for RNA Pol II

Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II) subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.

Rpb7 Can Interact with RNA Polymerase II and Support Transcription during Some Stresses Independently of Rpb4

ABSTRACT Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Δ, containing Pol IIΔ4) to grow above 30°C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability ofrpb4Δ cells to grow at 34°C (a relatively mild temperature stress) but not at higher temperatures. Overexpression ofRPB7 could also partially suppress the cold sensitivity ofrpb4Δ strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Δ cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIΔ4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIΔ4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIΔ4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIΔ4, enough to permit appropriate transcription also under some stress conditions.