The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization

STM1 IS A NOVEL REGULATOR OF MESSENGER RNA TRANSLATION AND DEGRADATION IN SACCHAROMYCES CEREVISIAE

In eukaryotes, regulation of translation and decay of messenger RNA are critical for fine-tuned control of gene expression. An important point of control is the key transition where mRNAs exit translation and assemble into a non-translating mRNP state that can accumulate in cytoplasmic granules such as P bodies and/or Stress granules. In the budding yeast Saccharomyces cerevisiae , the activators of decapping Dhh1 and Pat1 appear to promote the exit of mRNAs from translation. In my work, summarized below, I describe a new regulator of translation repression and mRNA degradation, Stm1, and its novel mode of action. First, I identified Stm1 as a novel regulator of translation repression and mRNA decay. Stm1 shows several genetic interactions with Pat1 and Dhh1, in a manner consistent with Stm1 promoting the function of Dhh1. This suggests that Stm1 has a role to play in translation repression and/or activation of mRNA decay. stm1 δ strains are defective in the degradation of a subset of mRNAs that include EDC1 and COX17 . These results strongly argue that Stm1 is a novel addition to the mRNA degradation machinery. Second, I have shown that Stm1, a known ribosome-associated protein, can bind and stall 80S ribosomes to repress translation and promote decay. Stm1 is able to repress translation and stall an 80S complex in vitro . Several mutations were identified in the protein, which link the in vitrophenotype to its biological functionin vivo. The analysis of different steps in translation reveals Stm1 functions in a novel manner to inhibit translation after the formation of an 80S complex. Since most of the regulation of translation is thought to happen at the stage of initiation, this study reveals a novel mode of translation regulation. These results also provide a direct and mechanistic link between ribosome function, inhibition of translation and the degradation of messenger RNAs.Embargo: Release after 7/9/201

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells

Stm1 modulates translation after 80S formation in Saccharomyces cerevisiae

The control of translation is a critical aspect of gene regulation. It is often inversely related to mRNA degradation and is typically controlled during initiation. The Stm1 protein in Saccharomyces cerevisiae has been shown to interact with ribosomes, affect the interaction of eEF3 with ribosomes, and promote the decapping of a subclass of mRNAs. We demonstrate that in vitro Stm1 inhibits translation after formation of an 80S complex. This suggests that Stm1 modulates translation and mRNA decapping by controlling translation elongation

Measurable residual disease monitoring for patients with acute myeloid leukemia following hematopoietic cell transplantation using error corrected hybrid capture next generation sequencing.

Improved systems for detection of measurable residual disease (MRD) in acute myeloid leukemia (AML) are urgently needed, however attempts to utilize broad-scale next-generation sequencing (NGS) panels to perform multi-gene surveillance in AML post-induction have been stymied by persistent premalignant mutation-bearing clones. We hypothesized that this technology may be more suitable for evaluation of fully engrafted patients following hematopoietic cell transplantation (HCT). To address this question, we developed a hybrid-capture NGS panel utilizing unique molecular identifiers (UMIs) to detect variants at 0.1% VAF or below across 22 genes frequently mutated in myeloid disorders and applied it to a retrospective sample set of blood and bone marrow DNA samples previously evaluated as negative for disease via standard-of-care short tandem repeat (STR)-based engraftment testing and hematopathology analysis in our laboratory. Of 30 patients who demonstrated trackable mutations in the 22 genes at eventual relapse by standard NGS analysis, we were able to definitively detect relapse-associated mutations in 18/30 (60%) at previously disease-negative timepoints collected 20-100 days prior to relapse date. MRD was detected in both bone marrow (15/28, 53.6%) and peripheral blood samples (9/18, 50%), while showing excellent technical specificity in our sample set. We also confirmed the disappearance of all MRD signal with increasing time prior to relapse (>100 days), indicating true clinical specificity, even using genes commonly associated with clonal hematopoiesis of indeterminate potential (CHIP). This study highlights the efficacy of a highly sensitive, NGS panel-based approach to early detection of relapse in AML and supports the clinical validity of extending MRD analysis across many genes in the post-transplant setting

Inflammation and Change in Body Weight with Antiretroviral Therapy Initiation in a Multinational Cohort of HIV-infected Adults.

BACKGROUND Both wasting and obesity are associated with inflammation, but the extent to which body weight changes influence inflammation in HIV is unknown. METHODS Among a random virologically suppressed participants of the PEARLS trial, inflammatory markers were measured at weeks 0, 24, and 48 post-antiretroviral therapy (ART). Associations between baseline and change in body mass index (BMI) and inflammation changes were assessed using random effects models. RESULTS Of 246 participants, 27% were overweight/obese (BMI≥25 kg/m(2)) and 8% were underweight (BMI<18.5 kg/m(2)) at baseline. After 48 weeks, 37% were overweight/obese and 3% were underweight. While many inflammatory markers decreased 48 weeks after ART in the overall group, the decrease in CRP was smaller in overweight/obese participants (p=0.01) and the decreases in both CRP (p=0.01) and IL-18 (p=0.02) were smaller in underweight participants. Each 1 unit gain in BMI among overweight/obese participants was associated with a 0.02 log10 increase in sCD14 (p=0.05), while each 1 unit BMI gain among underweight participants was associated with 9.32 mg/L decrease in CRP (p=0.001). CONCLUSION Being either overweight or underweight at ART initiation was associated with heightened systemic inflammation. While weight gain among overweight/obese persons predicted increased inflammation, weight gain among underweight persons predicted reduced inflammation

Inflammation and Change in Body Weight With Antiretroviral Therapy Initiation in a Multinational Cohort of HIV-Infected Adults.

BackgroundBoth wasting and obesity are associated with inflammation, but the extent to which body weight changes influence inflammation during human immunodeficiency virus infection is unknown.MethodsAmong a random virologically suppressed participants of the Prospective Evaluation of Antiretrovirals in Resource-Limited Settings trial, inflammatory markers were measured at weeks 0, 24, and 48 after antiretroviral therapy (ART) initiation. Associations between both baseline and change in body mass index (BMI; calculated as the weight in kilograms divided by the height in meters squared) and changes in inflammation markers were assessed using random effects models.ResultsOf 246 participants, 27% were overweight/obese (BMI, ≥ 25), and 8% were underweight (BMI &lt; 18.5) at baseline. After 48 weeks, 37% were overweight/obese, and 3% were underweight. While level of many inflammatory markers decreased 48 weeks after ART initiation in the overall group, the decrease in C-reactive protein (CRP) level was smaller in overweight/obese participants (P = .01), and the decreases in both CRP (P = .01) and interleukin 18 (P = .02) levels were smaller in underweight participants. Each 1-unit gain in BMI among overweight/obese participants was associated with a 0.02-log10 increase in soluble CD14 level (P = .05), while each 1-unit BMI gain among underweight participants was associated with a 9.32-mg/L decrease in CRP level (P = .001).ConclusionsBeing either overweight or underweight at ART initiation was associated with heightened systemic inflammation. While weight gain among overweight/obese persons predicted increased inflammation, weight gain among underweight persons predicted reduced inflammation