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RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

ABSTRACT: P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28b? (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.

SUBMITTER: Fernandes N

PROVIDER: S-EPMC7293044 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

Fernandes Nikita N Buchan J Ross JR

Nucleic acids research 20200601 11

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B ...[more]

PMID: 32396167

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Project description:Cis-regulatory elements (CREs) dictate spatiotemporal expression and tissue specificity of proximal genes. However, when in a transgenic state, many of them become highly vulnerable to RNA-Directed DNA Methylation (RdDM) that is often transcriptionally deleterious and biologically detrimental. This transgene-specific RdDM vulnerability suggests the existence of anti-RdDM elements (AREs) to defend CREs against de novo methylation in vivo. In this work, we identify such an ARE at the Arabidopsis AGAMOUS (AG) locus, which includes a physically separated enhancer and promoter, both of which are highly vulnerable to transgene silencing. We demonstrate that this ARE effectively represses RdDM activity at its cognate and heterologous Cis-regulatory elements (CREs) control gene expression amplitude, tissue specificity and timing, whereby they directly impact phenotypic variation. However, in plants, CREs frequently become prone to RNA-directed DNA methylation (RdDM) when in a transgenic state (particularly in a tandem-repeat configuration), and the mechanism driving this phenomenon is currently unknown. In this study, we demonstrate that three of six flower-specific CREs, including the AGAMOUS enhancer (AGe), undergo transcriptional silencing when in a transgenic state, leading to the production of mutant flowers in 28-44% of transgenic lines. This silencing is dependent on the transgene self-transcribed non-coding RNAs (ncRNAs) that feed into biogenesis of 24-nt small interfering RNAs (siRNAs) and the canonical RdDM pathway, but not RNAs transcribed by the RNA polymerase II promoter. Strikingly, this ncRNA-dependent, siRNA-driven RdDM is prone to the enhancing action of an adjacent CaMV35S enhancer (35Se), which was shown to promiscuously interact with, and ectopically activate, numerous CREs including the AGe. This enhancement corresponded with an increase in ncRNAs specifically transcribed from the antisense strand of the transgene. We further demonstrate that the same 35Se inserted approximately 3.5 kb upstream of the native AGe within a genome promiscuously interacts with and activating de novo RdDM activity in the AGe, resulting in a mutant flower. These findings provide the first evidence that promiscuous CRE-CRE interactions serve as a causative force driving RdDM/silencing, and novel insight into the mechanism underlying the methylation-vulnerablity of the CRE transgenes, duplicated genes and repetitive genomic regions rich in transposon elements, where promiscuous CRE-CRE interactions are common.

Dataset Information

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

Publications

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

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