Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

Abstract

RNA-binding protein Sbp1 facilitates the decapping pathway in mRNA metabolism and inhibits global mRNA translation by an unclear mechanism. Here we report molecular interactions responsible for Sbp1-mediated translation inhibition of mRNA encoding the polyadenosine-binding protein (Pab1), an essential translation factor that stimulates mRNA translation and inhibits mRNA decapping in eukaryotic cells. We demonstrate that the two distal RRMs of Sbp1 bind to the poly(A) sequence in the 5 ′ ′ ′ ′ ′ UTR of the Pab1 mRNA specifically and cooperatively while the central RGG domain of the protein interacts directly with Pab1. Furthermore, methylation of arginines in the RGG domain abolishes the protein-protein interaction and the inhibitory effect of Sbp1 on translation initiation of Pab1 mRNA. Based on these results, the underlying mechanism for Sbp1-specific translational regulation is proposed. The functional differences of Sbp1 and RGG repeats alone on transcript-specific translation were observed, and a comparison of the results suggests the importance of remodeling the 5 ′ ′ ′ ′ ′ UTR by RNA-binding proteins in mRNA translation.

Arginine methylation augments Sbp1 function in translation repression and decapping

“…Recently Sbp1 was reported to bind Pab1 mRNA in the 5′ UTR region and repress its translation in vitro . Interestingly this report shows that Sbp1 protein (methylated in E. coli ) represses translation of luciferase mRNA weaker than unmethylated Sbp1.…”

“…As a control, wild‐type untagged strain of BY4741 was used. Previous reports indicate that Sbp1 tagged with GST and GFP interacts with other binding partners and localize to RNA granules respectively indicating that such tags do not alter its activity . The pull‐downs were probed with mono‐methyl arginine specific antibody (CST, cat#8711) followed by stripping and re‐probing the same blot with anti‐GST antibody (CST, cat#2624).…”

Conserved heterodimeric GTPase Rbg1/Tma46 promotes efficient translation in eukaryotic cells

“…The binding constants of tRNA with purified Rbg1 and Tma46 were measured using a filter binding assay as described 47 with minor modifications. 32 P-labeled tRNA 48,49 was incubated with the Rbg1/Tma46 complex in binding buffer (25 mM HEPES-KOH, pH 7.6, 2.5 mM Mg(OAc)2, 80 mM KOAc, 2 mM DTT, 0.5 mM GDPCP•Mg 2+ ) at 30 ˚C for 10 minutes. The reaction solution was subsequently filtered through two membranes simultaneously, the first being a nitrocellulose membrane (GE Healthcare Amersham) and the second being a Nytran Supercharge membrane (Whatman).…”

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering

“…In vivo and in vitro studies have identified several Hmt1 substrates [36–38]. Moreover, Hmt1-mediated methylation has been shown to enhance the function of its substrates in multiple pathways, including chromatin remodeling and transcription [39–41], translation and ribosome biogenesis [42–45], and posttranscriptional regulation [46–49]. We selected the representative proteins Npl3, Rps2, Sbp1, and Snf2 from among these pathways and generated corresponding deletion or hypomorphic mutants (in cases when mutant haploids died or had severe growth defects) and investigated noise levels among these mutants.…”