Dataset Information

Cross-species functionality of pararetroviral elements driving ribosome shunting.

ABSTRACT:

Background

Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5'-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.

Methodology/principal findings

Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.

Conclusions/significance

Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism. We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.

SUBMITTER: Pooggin MM

PROVIDER: S-EPMC2241666 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Publications

Cross-species functionality of pararetroviral elements driving ribosome shunting.

Pooggin Mikhail M MM Fütterer Johannes J Hohn Thomas T

PloS one 20080220 2

<h4>Background</h4>Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5'-proximal sORF terminating near the stem. In CaMV, mutations knoc ...[more]

PMID: 18286203

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Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC-seq and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: ATAC-seq libraries were prepared from adipose, liver, muscle, and islets as described in [Corces et al., 2016 (Nature Genetics) with slight modifications. Adipose, liver, and muscle samples were dissociated into single cell suspensions using MACS Miltenyi Biotec Adipose Tissue (130-105-808), Liver (130-105-807), and Skeletal Muscle (130-098-305) Dissociation Kits. Dissociated cells were counted and 50,000 cells were resuspended in freezing media (10% DMSO, 50% FBS, 40% RPMI) and frozen at -80°C until cells were thawed and washed in cold PBS, and incubated in ATAC lysis buffer. Islets were isolated from total pancreas as described in [Simonett et al., 2021 (JCI)], and 50 islets were rested overnight in RPMI in 37°C incubator. The following day islets were washed in cold PBS, incubated in ATAC lysis buffer, and triturated with 25-gauge needle and centrifuged to generate a crude nuclei pellet, and washed in ATAC lysis buffer. Prepared nuclei from all tissues were transposed using the Tagment DNA TDE1 Enzyme and Buffer kit (Illumina) with the addition of 0.01% digitonin (1x TDE for liver, muscle; 2x TDE for adipose). Transposed DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo Research), amplified with 11 cycles of PCR, and cleaned using KAPA Pure Beads (Roche). All ATAC-seq libraries were sequenced at 2 x 100 bp using Illumina NovaSeq 6000. 1. Corces M.R., Buenrostro J.D., Wu B., Greenside P.G., Chan S.M., Koenig J.L., Snyder M.P., Pritchard J.K., Kundaje A., Greenleaf W.J. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016;48:1193–1203. https://www.nature.com/articles/ng.3646 2. Simonett S.P., Shin S., Herring J.A., Bacher R., Smith L.A., Dong C., Rabaglia M.E., Stapleton D.S., Schueler K.L., Choi J., Bernstein M.N., Turkewitz D.R., Perez-Cervantes C., Spaeth J., Stein R., Tessem J.S., Kendziorski C., Keleş S., Moskowitz I.P., Keller M.P., Attie A.D. Identification of direct transcriptional targets of NFATC2 that promote beta-cell proliferation. J Clin Invest. 2021;131(21):e144833. https://doi.org/10.1172/JCI144833.

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Dataset Information

Cross-species functionality of pararetroviral elements driving ribosome shunting.

Background

Methodology/principal findings

Conclusions/significance

Publications

Cross-species functionality of pararetroviral elements driving ribosome shunting.

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