Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export

Abstract

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5= end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae. Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6

“…Yeast is an ideal research model for transcriptome research because they exhibit most of the cellular complexity present in eukaryotes and have relatively compact, accessible genomes. However, while the interconnected transcriptional circuit has been studied and accepted by many yeast labs 2,[10][11][12][13][14][15] , these studies usually target aspects of the transcriptional regulation and produce separate results that contribute to the overall transcriptional model. Moreover, different strains, experimental conditions and batches are used by different labs and there are few examples of experimental datasets where all these layers have been measured on exactly the same samples.…”

The deubiquitylase Ubp15 couples transcription to mRNA export

“…Interestingly, all those interactors were less associated with Mex67 in ubp15Δ cells, suggesting that the ubiquitylation of Mex67 negatively regulates its association with these factors. Strikingly, these proteins include all four subunits of the THO complex (Hpr1, Tho2, Mft1, and Thp2) and both subunits of the heterodimeric cap-binding complex (CBC; Sto1 and Cbc2) known to recruit THO/TREX to the 5’-cap of nascent pre-mRNAs ( Figure 5D ) in yeast and humans ( Cheng et al, 2006 ; Sen et al, 2019 ; Viphakone et al, 2019 ). We noted also that several subunits of the NPC, including the nuclear basket protein Mlp1, showed a slightly increased association with Mex67 in ubp15Δ cells compared to WT cells (although below our significance threshold) ( Figure 5D ).…”

“…Then, Hpr1 binds Sub2 on nascent transcripts and is implicated in the early recruitment of Mex67, an interaction mediated by a ubiquitin-dependent process ( Gwizdek et al, 2005 ; Gwizdek et al, 2006 ; Zenklusen et al, 2002 ). Yra1 is also co-transcriptionally recruited, notably by the cap binding complex (CBC) ( Cheng et al, 2006 ; Sen et al, 2019 ; Viphakone et al, 2019 ) and transferred to Sub2 ( Johnson et al, 2009 ; Johnson et al, 2011 ). Later, TREX-2 (Sac3, Thp1, Sus1, Cdc31, and Sem1) interacts with the nuclear side of the NPC and participates in the coordination between transcription and mRNA export ( Cabal et al, 2006 ; Fischer et al, 2004 ; Jani et al, 2009 ; Rodríguez-Navarro et al, 2004 ).…”

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function

“…Isolation of total and cytoplasmic RNAs. Total RNA was prepared from yeast cell culture as done previously (18,30,99). Briefly, 10 ml yeast culture was harvested and then suspended in 100 l RNA preparation buffer (500 mM NaCl, 200 mM Tris-HCl, 100 mM Na 2 EDTA, and 1% SDS) along with 100 l phenol-chloroform-isoamyl alcohol and a 100-l volume equivalent of glass beads (acid washed; Sigma).…”

“…Cytoplasmic RNA was prepared from yeast cells as described previously (18,30,99). Briefly, harvested yeast cells from a 10-ml culture were suspended in 400 l sorbitol solution (0.9 M sorbitol, 0.1 M EDTA, and 14 mM ␤-mercaptoethanol) and then were incubated with 20 l Zymolyase (10 mg/ml) for 25 min at 37°C followed by centrifugation for 5 to 10 s. The supernatant was carefully removed, and the spheroplast was gently suspended in 100 l RNA preparation buffer along with a 20-l volume equivalent of glass beads for immediate cytoplasmic fractionation with mild vortexing (around 15 s on the VWR minivortexer with a low speed of 5).…”