Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1⌬ null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.The eukaryotic 80S ribosome, consisting of small (40S) and large (60S) subunits, is the catalytic and regulatory macromolecular complex responsible for the decoding of mRNA into polypeptides. Together, the small and large ribosomal subunits contain the 18S, 28S (25S in yeast), 5.8S, and 5S rRNAs along with a large number of proteins. The structure and function of the ribosome have been extensively studied for decades, and a 15 Å cryoelectron microscopic map of the yeast ribosome is available (51). While the catalytic activity of the ribosome's 28S rRNA in peptide bond formation is well established, the function and regulatory activity of the ribosomal proteins are still largely unknown (12).Advanced mass spectrometry (MS) analysis has facilitated the identification of novel ribosomal proteins. In a proteomic screen of the Saccharomyces cerevisiae 40S, 60S, and 80S components, we identified a novel component of the 40S and 80S subunits, Asc1p (ASC1, YMR116C, CPC2, BEL1), which remains associated with the small subunit in the presence of 1 M KCl (34). Under these stringent conditions, transient translation factors and ribosome biogenesis factors present in lower salt concentrations are shed from the 40S component. In these experiments, Asc1p was present at a concentration equimolar to that of the other ribosomal proteins (34). By established criteria for defining ribosomal proteins, Asc1p can be classified as a novel core 40S ribosomal component (31). We also demonstrated that RACK1 (receptor for activated C kinase 1), a protein with 52% sequence identity to Asc1p, is localized to the 40S and 80S components and polysomes in human cells (34). These observations have been confirmed by several other studies (2, 4, 6, 26, 50).RACK1 was originally identified as a protein with sequence similarity to the guanine nucleotide-binding protein  subunit and other proteins containing Trp-Asp (WD) repeat domains (20). RACK1 was later shown to associate both in vitro and in vivo with activated prote...
Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.
Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens.
BackgroundThe Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some.Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations.ResultsWe found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes.Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference.Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays.ConclusionsThe greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species.The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1928-z) contains supplementary material, which is available to authorized users.
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