SENSE Total RNA-Seq Publications

A hypomorphic Stip1 allele reveals the requirement for chaperone networks in mouse development and aging

Rachel Lackie, Marilene H Lopes, Sali M.K. Farhan, Abdul Razzaq, Gilli Moshitzky, Mariana B Prado, Flavio H Beraldo, Andrzej Maciejewski, Robert Gros, Jue Fan, Wing-Yiu Choy, David Greenberg, Vilma R Martins, Martin Duennwald, Hermona Soreq, Vania F Prado, Marco A. M. Prado

biorXiv, doi:10.1101/258673

The chaperone machinery is well conserved from yeast to mammals, however our knowledge of their impact on mammalian physiology is lagging. Stress-inducible phosphoprotein-1 (STI1; STIP1; Hop) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90 via three tetratricopeptide repeat (TPR) domains, of which TPR1 and TPR2B may be redundant in yeast. In-depth analysis of human datasets indicated that STI1 belongs to a set of co-chaperones that is essential in humans and that the TPR1 domain is evolutionarily conserved, suggesting that in mammals it may be required for optimal STI1 activity in vivo. We generated mice with a hypomorphic Stip1 allele lacking the TPR1 domain. While these mice are viable, they presented decreased levels of Hsp90 client proteins and co-chaperones, suggesting profound dysregulation of chaperone networks. We used this hypomorphic STI1 mutant mouse line to investigate the requirement of STI1-mediated regulation of chaperone networks in mouse physiology. Embryonic cell pluripotency was severely affected by decreased STI1 activity, contributing to the abnormal development in these mice. Moreover, adult TPR1-deprived STI1 mice presented age-related hippocampal neurodegeneration, resulting in compromised memory recall. Our findings reveal a requirement for optimal regulation of chaperone networks and their clients during development and strict dependence on full STI1 activity for healthy neuronal aging. These experiments demonstrate the unique experimental power of using hypomorphic alleles to reveal how chaperone networks regulate mammalian physiology.

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The baculovirus insect cell expression system has become a firmly established production platform in biotechnology. Various complex proteins, multi-subunit particles including veterinary and human vaccines are manufactured with this system on a commercial scale. Apart from baculovirus infected Spodoptera frugiperda (Sf9) cells, the Trichoplusia ni(HighFive) cell line is alternatively used as host organism. In this study, we explored the protein production capabilities of Tnms42 insect cells, a new derivative of HighFive, which is free of latent nodavirus infection. As a model system, a cytosolic (mCherry) and a secreted (hemagglutinin) protein were overexpressed in Tnms42 cells. The response of the host cells was followed in a time course experiment over the infection cycle by comparative transcriptome analysis (RNA-seq). As expected, the baculovirus infection per se had a massive impact on the host cell transcriptome, which was observed by the huge total number of differentially expressed transcripts (>14,000). Despite this severe overall cellular reaction, a specific response could be clearly attributed to the overexpression of secreted hemagglutinin, revealing limits in the secretory capacity of the host cell. About 400 significantly regulated transcripts were identified and assigned to biochemical pathways and gene ontology (GO) categories, all related to protein processing, folding and response to unfolded protein. The identification of relevant target genes will serve to design specific virus engineering concepts for improving the yield of proteins that are dependent on the secretory pathway.

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Extensive studies have suggested that most miRNA functions are executed through complex miRNA-target interaction networks, and such networks function semiredundantly with other regulatory systems to shape gene expression dynamics for proper physiological functions. We found that knocking down vgln-1, which encodes a conserved RNA-binding protein associated with diverse functions, causes severe larval arrest at the early L1 stage in animals with compromised miRISC functions (an ain-2/GW182 mutant). Through an enhancer screen, we identified five specific miRNAs, and miRNA families, that act semiredundantly with VGLN-1 to regulate larval development. By RIP-Seq analysis, we identified mRNAs that are directly bound by VGLN-1, and highly enriched for miRNA binding sites, leading to a hypothesis that VGLN-1 may share common targets with miRNAs to regulate gene expression dynamics for development.

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Epistatic and allelic interactions control expression of ribosomal RNA gene clusters in Arabidopsis thaliana

Fernando A. Rabanal, Terezie Mandáková, Luz M. Soto-Jiménez, Robert Greenhalgh, David L. Parrott, Stefan Lutzmayer, Joshua G. Steffen, Viktoria Nizhynska, Richard Mott, Martin A. Lysak, Richard M. Clark and Magnus Nordborg

Genome Biology 2017, doi:10.1186/s13059-017-1209-z

Ribosomal RNA (rRNA) accounts for the majority of the RNA in eukaryotic cells, and is encoded by hundreds to thousands of nearly identical gene copies, only a subset of which are active at any given time. In Arabidopsis thaliana, 45S rRNA genes are found in two large ribosomal DNA (rDNA) clusters and little is known about the contribution of each to the overall transcription pattern in the species.

By taking advantage of genome sequencing data from the 1001 Genomes Consortium, we characterize rRNA gene sequence variation within and among accessions. Notably, variation is not restricted to the pre-rRNA sequences removed during processing, but it is also present within the highly conserved ribosomal subunits. Through linkage mapping we assign these variants to a particular rDNA cluster unambiguously and use them as reporters of rDNA cluster-specific expression. We demonstrate that rDNA cluster-usage varies greatly among accessions and that rDNA cluster-specific expression and silencing is controlled via genetic interactions between entire rDNA cluster haplotypes (alleles).

We show that rRNA gene cluster expression is controlled via complex epistatic and allelic interactions between rDNA haplotypes that apparently regulate the entire rRNA gene cluster. Furthermore, the sequence polymorphism we discovered implies that the pool of rRNA in a cell may be heterogeneous, which could have functional consequences.

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