PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.231530v1?rss=1

Authors: Liu, J., Hu, X., Bao, K., Kim, J.-K., Jia, S., Qiao, F.

Abstract:

Shelterin is a six-proteins complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.01.230755v1?rss=1

Authors: Zhang, J., Guo, P., Yu, X., Pouchnik, D., Firrman, J., Wei, H., Sang, N., Li, D., Herzog, R., Diao, Y., Xiao, W.

Abstract:

Recombinant AAV (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Nevertheless, current rAAV vectors administered to patients often contain non-vector related DNA contaminants. Here, we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production. In addition to the sub-vector genomic size particles containing incomplete AAV genomes, our results found that rAAV preparations were contaminated with multiple categories of subgenomic particles with either snapback genomes or vector genomes with deletions in the mid regions. Through CRISPR and restriction enzyme-based in vivo and in vitro modeling, we identified that the main mechanism leading to the formation of non-canonical genome particles occurred through nonhomologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks that were generated by the host cell/environment. The results of this study advance our understanding of AAV vectors and provide new clues on improving vector efficiency and safety profile for use in human gene therapy.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228734v1?rss=1

Authors: Smyth, L. J., Kilner, J., Nair, V., Liu, H., Brennan, E., Kerr, K., Sandholm, N., Cole, J., Dahlstrom, E., Syreeni, A., Salem, R. M., Nelson, R. G., Looker, H. C., Wooster, C., Anderson, K., McKay, G., Kee, F., Young, I., NICOLA Collaborative Team,, Warren 3 and Genetics of Kidneys in Diabetes (GoKinD) Study Group,, Andrews, D., Forsblom, C., Hirschhorn, J. N., Godson, C., Groop, P.-H., Maxwell, A. P., Susztak, K., Kretzler, M., Florez, J. C., McKnight, A. J., GENIE consortium

Abstract:

A subset of individuals with type 1 diabetes mellitus (T1DM) are predisposed to developing diabetic kidney disease (DKD), which is the most common cause globally of end-stage kidney disease (ESKD). Emerging evidence suggests epigenetic changes in DNA methylation may have a causal role in both T1DM and DKD. The aim of this investigation was to assess differences in blood-derived DNA methylation patterns between individuals with T1DM-ESKD and individuals with long-duration T1DM but no evidence of kidney disease upon repeated testing. Blood-derived DNA from individuals (107 cases, 253 controls and 14 experimental controls) were bisulphite treated before DNA methylation patterns from both groups were generated and analysed using Illumina's Infinium MethylationEPIC BeadChip arrays (n=862,927 sites). Differentially methylated CpG sites (dmCpGs) were identified (false discovery rate adjusted p[≤]x10-8 and fold change {+/-}2) by comparing methylation levels between ESKD cases and T1DM controls at single site resolution. Gene annotation and functionality was investigated to enrich and rank methylated regions associated with ESKD in T1DM. Top-ranked genes within which several dmCpGs were located and supported by in silico functional data, and replication where possible, include; AFF3, ARID5B, CUX1, ELMO1, FKBP5, HDAC4, ITGAL, LY9, PIM1, RUNX3, SEPTIN9, and UPF3A. Top-ranked enrichment pathways included pathways in cancer, TGF-{beta} signalling and Th17 cell differentiation. Epigenetic alterations provide a dynamic link between an individual's genetic background and their environmental exposures. This robust evaluation of DNA methylation in carefully phenotyped individuals, has identified biomarkers associated with ESKD, revealing several genes and implicated key pathways associated with ESKD in individuals with T1DM.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.229070v1?rss=1

Authors: Ballou, E. R., Cook, A. G., Wallace, E. W. J.

Abstract:

The RNase II family of 3'-5' exoribonucleases are present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like "pseudonucleases". These function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. Here, we show how these pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. The N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the non-nuclease function require this region. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid "titan" growth stages and yet retains an active site sequence signature. We propose that that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, while the RNase activity was lost repeatedly in independent lineages.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226936v1?rss=1

Authors: Plassart, L., Shayan, R., Montellese, C., Rinaldi, D., Larburu, N., Pichereaux, C., Lebaron, S., O'Donohue, M.-F., Kutay, U., Marcoux, J., Gleizes, P.-E., Plisson-Chastang, C.

Abstract:

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.230284v1?rss=1

Authors: Beagrie, R. A., Thieme, C. J., Annunziatella, C., Baugher, C., Zhang, Y., Schueler, M., Kramer, D. C., Chiariello, A. M., Bianco, S., Kukalev, A., Li, Y., Kempfer, R., Scialdone, A., Welch, L. A., Nicodemi, M., Pombo, A.

Abstract:

Technologies for measuring 3D genome topology are increasingly important for studying mechanisms of gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of Genome Architecture Mapping (GAM), a ligation-free technique to map chromatin contacts genome-wide. We perform a detailed comparison of contacts obtained by multiplex-GAM and Hi-C using mouse embryonic stem (mES) cells. We find that both methods detect similar topologically associating domains (TADs). However, when examining the strongest contacts detected by either method, we find that only one third of these are shared. The strongest contacts specifically found in GAM often involve "active" regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain "inactive" regions. Our work shows that active genomic regions are involved in extensive complex contacts that currently go under-estimated in genome-wide ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.230870v1?rss=1

Authors: Blume, C., Jackson, C. L., Spalluto, C. M., Legebeke, J., Nazlamova, L. A., Conforti, F., Perotin-Collard, J.-M., Frank, M., Crispin, M., Coles, J., Thompson, J., Ridley, R. A., Dean, L. S., Loxham, M., Azim, A., Tariq, K., Johnston, D. A., Skipp, P. J., Djukanovic, R., Baralle, D., McCormick, C. J., Davies, D. E., Lucas, J. S., Wheway, G., Mennella, V.

Abstract:

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear. Until now ACE2 was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. Short ACE2 is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether our data are consistent with a model where short ACE2 may influence host susceptibility to SARS-CoV-2 infection.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.231225v1?rss=1

Authors: Santharam, M. A., Shukla, A., Ihsan, A. U., Cloutier, M., Levesque, D., Ramanathan, S., Boisvert, F.-M., Ilangumaran, S.

Abstract:

Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma (HCC) and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor MET, the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, may dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis. Notably, the expression of UBE2D ubiquitin conjugating enzymes, which are implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.229351v1?rss=1

Authors: Cao, J., Routh, A., Kuyumcu-Martinez, M. N.

Abstract:

Alternative splicing (AS) increases the proteome variety by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full length Tpm1 isoforms composed of distinct exons with specific linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. Nanopore sequencing combined with polyA sequencing revealed that the RNA binding protein RBFOX2 controls AS of muscle specific Tpm1 isoforms and expression of TPM1 protein via terminal exon splicing impacting its polyadenylation. RBFOX2 regulates Tpm1 AS antagonistically to PTBP1. In sum, we defined full length Tpm1isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of muscle specific isoforms of Tpm1 by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle enriched genes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227728v1?rss=1

Authors: Davis, B. K.

Abstract:

Sequence homology in pre-divergence tRNA species revealed cofactor/adaptors cognate for 16 amino acids derived from oxaloacetate, pyruvate, phosphoglycerate, or phosphoenolpyruvate were related. Synthesis path-distances of these amino acids correlated with phylogenetic depth, reflecting relative residue frequency in pre-divergence sequences. Both metrics were thus aligned in the four sub-families of the Aspartate family, and misaligned in the small Glutamate family; a functional difference was noted and seen to parallel synthetase duality. Amino acid synthetic order, based on path-distances, indicate NH4+ fixer amino acids, Asp1, Asn2, and homologues, Glu1, Gln2, formed the first code. Together with a termination signal, they acquired all four triplet 4-sets in the XAN column (X, 5-prime coding site; N, any 3-prime-base). An invariant mid-A conformed with pre-code translation on a poly(A) template by a ratchet-equipped ribosome resulting in random, polyanionic polypeptides. Code expansion occurred in a compact (mutation minimizing) column-wise pattern, (XAN)to XCN to XGN to XUN; with increasing mean path-distance, (1.5) to 4 to 5 to 7 steps; amino acid side-chain hydro-phobicity, (+6.6)to -0.8 to -1.5 to-3.2 kcal/mol; codon:anticodon H bond enthalpy (selection for bond-strength), (-12.5)to -17.5 to -15.5 to -14.5 kcal/mol; and precursor-specific 5-prime-base, A, oxaloacetate, G, pyruvate/ oxalo-acetate, U, phosphoglycerate/oxaloacetate, C, oxoglutarate, forming horizontal code domains. Codon bias evidence corroborated the XCN to XGN step in expansion, and revealed row GNN coevolved with ANN, on correction for overprinting. Extended surface-attachment (Fajan-Paneth principle) by pro-Fd[5] and bilayer partitioning by H+ ATPase proteolipid-h1 subunit implicated expansion phase proteins in driving increases in side-chain hydrophobicity during code expansion. 3-prime-Base recruitment in pre-assigned codon boxes added six long (9- to 14-step) path amino acid, bearing a basic, or cyclic, side-chain; 3 of 4 polar, post-expansion amino acids acquired polar cluster NAN codons and 2 of 3 non-polar (Ile7 included) acquired non-polar cluster NUN codons, yieldng a split-box pair homology of p = 5.4x10-3. All eight overprinted codon boxes (GAYR for Asp1, Glu1 included) exhibit weak codon: anti-codon H-bond enthalpy, -14 kcal/mol or higher, in three of six distinct code enthalpy states.

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