EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization.

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation

Mining literature for a comprehensive pathway analysis: A case study for retrieval of homocysteine related genes for genetic and epigenetic studies

<p>Abstract</p> <p>Homocysteine is an independent risk factor for cardiovascular diseases. It is also known to be associated with a variety of complex disorders. While there are a large number of independent studies implicating homocysteine in isolated pathways, the mechanism of homocysteine induced adverse effects are not clear. Homocysteine-induced modulation of gene expression through alteration of methylation status or by hitherto unknown mechanisms is predicted to lead to several pathological conditions either directly or indirectly. In the present manuscript, using literature mining approach, we have identified the genes that are modulated directly or indirectly by an elevated level of homocysteine. These genes were then placed in appropriate pathways in an attempt to understand the molecular basis of homocysteine induced complex disorders and to provide a resource for selection of genes for polymorphism screening and analysis of mutations as well as epigenetic modifications in relation to hyperhomocysteinemia. We have identified 135 genes in 1137 abstracts that either modulate the levels of homocysteine or are modulated by elevated levels of homocysteine. Mapping the genes to their respective pathways revealed that an elevated level of homocysteine leads to the atherosclerosis either by directly affecting lipid metabolism and transport or via oxidative stress and/or Endoplasmic Reticulum (ER) stress. Elevated levels of homocysteine also decreases the bioavailability of nitric oxide and modulates the levels of other metabolites including S-adenosyl methionine and S-adenosyl homocysteine which may result in cardiovascular or neurological disorders. The ER stress emerges as the common pathway that relates to apoptosis, atherosclerosis and neurological disorders and is modulated by levels of homocysteine. The comprehensive network collated has lead to the identification of genes that are modulated by homocysteine indicating that homocysteine exerts its effect not only through modulating the substrate levels for various catalytic processes but also through regulation of expression of genes involved in complex diseases.</p

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity

Single nucleotide polymorphisms in homocysteine metabolism pathway genes: association of CHDH A119C and MTHFR C677T with hyperhomocysteinemia

Background—An elevated level of homocysteine (hyperhomocysteinemia) has been implicated as an independent risk factor for cardiovascular diseases. Deficiency of dietary factors like vitamin B12, folate and/or genetic variations can cause hyperhomocysteinemia. The prevalence of hyperhomocysteinemia in Indian population is likely to be high since a majority of Indians adhere to a vegetarian diet, deficient in vitamin B12. In the background of vitamin B12 deficiency, variations in genes involved in homocysteine metabolism might have a greater impact on homocysteine levels. Methods and Results—We genotyped 44 non-synonymous single nucleotide polymorphisms (nsSNPs) from 11 genes involved in homocysteine metabolism and found only 14 to be polymorphic. These 14 nsSNPs were genotyped in 546 individuals recruited from a tertiary care centre in New Delhi, India and it was found that choline dehydrogenase (CHDH A119C) and methylenetetrahydrofolate reductase (MTHFR C677T) were significantly associated with plasma total homocysteine levels (p=0.009 and p=0.001 respectively). These 2 SNPs were further genotyped in 330 individuals recruited from the same centre and the association remained significant even after increasing the sample size. Further, we found the possibility of a significant interaction between vegetarian diet and the two polymorphisms that could explain the variation of homocysteine levels. We also genotyped all the polymorphic nsSNPs in apparently healthy individuals recruited from 24 different sub-populations (based on their linguistic lineage) spread across the country to determine their basal frequencies. The frequencies of these SNPs varied significantly between linguistic groups. Conclusion—Vegetarian diet along with CHDH A119C and MTHFR C677T play an important role in modulating the homocysteine levels in Indian population

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization

Germline mis-sense mutations affecting a single BRCA2 allele predispose humans to cancer. Here, we identify a protein-targeting mechanism disrupted by the cancer-associated mutation, BRCA2(D2723H) that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mis-localization of mutant BRCA2 inhibits the nuclear retention of RAD51, by exposing a similar NES in RAD51 usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Interestingly, BRCA2(D2723H) decreases RAD51 nuclear retention even when wildtype BRCA2 is present. Our findings suggest a mechanism for regulation of the nucleo-cytoplasmic distribution of BRCA2 and RAD51, and for its impairment by a heterozygous disease-associated mutation