Dataset Information

AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

ABSTRACT: 3' untranslated regions (3' UTRs) and particularly long 3' UTRs have been shown to act as a new class of post-transcriptional regulatory element. We previously reported that hmsT mRNA stability is negatively regulated by the 3' UTR of hmsT in Yersinia pestis. To investigate more general effects of 3' UTRs in Y. pestis, we selected 15 genes potentially possessing long 3' UTRs with different AU content and constructed their 3' UTR deletion mutants. Deletion of AU-rich 3' UTRs increased mRNA levels, whereas deletion of 3' UTRs with normal AU content resulted in slight or no changes in the mRNA level. In addition, we found that PNPase was important for 3' UTR-mediated mRNA decay when the transcriptional terminator was Rho-dependent. Finally, we showed that ribosomes promote mRNA stability when bound to a 3' UTR. Our findings suggest that functional 3' UTRs might be broadly distributed in bacteria and their novel regulatory mechanisms require further investigation.

SUBMITTER: Zhao JP

PROVIDER: S-EPMC6299119 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

Zhao Ju-Ping JP Zhu Hui H Guo Xiao-Peng XP Sun Yi-Cheng YC

Frontiers in microbiology 20181212

3' untranslated regions (3' UTRs) and particularly long 3' UTRs have been shown to act as a new class of post-transcriptional regulatory element. We previously reported that hmsT mRNA stability is negatively regulated by the 3' UTR of hmsT in Yersinia pestis. To investigate more general effects of 3' UTRs in Y. pestis, we selected 15 genes potentially possessing long 3' UTRs with different AU content and constructed their 3' UTR deletion mutants. Deletion of AU-rich 3 ...[more]

PMID: 30619162

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Project description:Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3'UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing "free" target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks.

Project description:The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

Dataset Information

AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

Publications

AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

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