Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex

Abstract

The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs. In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex. We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation. Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths. By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes. We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1.

Fig. 1.

Gbp2 and Hrb1 are components of the TREX complex. (A) TAP-tagged Gbp2 (lanes 3 and 4) and Hrb1 (lanes 5 and 6) were purified from Saccharomyces cerevisiae, and copurifying proteins were identified by mass spectrometry. For comparison, purification of TAP-tagged Hpr1 (lane 1) and Sub2 (lane 2) are shown. Tagged proteins are marked with an asterisk. TREX complex components are indicated by a filled circle. From to top bottom: Tho2, Hpr1, Hrb1 (only visible in lane 1), Sub2, Mft1, Gbp2 (all three proteins comigrate), Tex1, Thp2, and Yra1. Yeast lysates were treated with RNase A before purification of Gbp2 (lane 4) and Hrb1 (lane 6). (B) Npl3 does not associate with the TREX complex. TAP-tagged Npl3 was purified from yeast, and copurifying proteins were identified by mass spectrometry (lane 2). For comparison, a purification of Hrb1-TAP is shown (lane 1). Tagged proteins are indicated by an asterisk. Copurifying proteins are numbered and correspond as follows: 1, Kem1; 2, Snt1; 3, Tif-4F; 4, Rat1 and Tif-4F; 5, Sto1/Cbp80; 6, Ssb1, Ded1, and Pab1; and 7, Gbp2. Common contaminants are indicated by a filled square and correspond to a porin from Escherichia coli and ribosomal proteins. (C) Gpb2 and Hrb1 are not necessary for the stability of the TREX complex. Purification of TAP-tagged Sub2 (lanes 1 and 2) and Hpr1 (lanes 3 and 4) from wild-type yeast cells (lanes 1 and 3) and cells lacking both Gbp2 and Hrb1 (lanes 2 and 4). The tagged proteins are indicated by an asterisk, and TREX complex components are indicated by filled circles, as in A. The leftmost lanes show molecular weight markers (M).

Fig. 2.

Gbp2 and Hrb1 associate with actively transcribed genes. Gbp2 and Hrb1 associate with the constitutively expressed PMA1 gene. ChIP experiments were performed by using nontagged (no tag), Hrb1-myc-tagged (Hrb1), and Gbp2-myc-tagged (Gbp2) strains in the absence (–) or presence (+) of RNase A. Coimmunoprecipitated DNA was analyzed by PCR using primers specific for the 5′, middle (M), and 3′ regions of the PMA1 gene and a nontranscribed region as negative control (Ref.). As a positive control, PCRs were performed by using whole-cell extract as template (total).

Fig. 3.

Gbp2 and Hrb1 bind to transcripts of several genes. For RNA-IP analysis, RNA coimmunoprecipitated with myc-tagged Gbp2 or Hrb1 was reverse-transcribed and quantified by PCR using specific primers to regions of the GAL1, PMA1, HYP2, RPS5, DBP2, and ACT1 genes. As controls, immunoprecipitation with a control antibody (control) and RT-PCRs with whole-cell extracts as template (total) are shown.

Fig. 4.

Gbp2 and Hrb1 are recruited to the transcription complex by their interaction with Ctk1. (A) Synthetic lethal interaction between MFT1 and CTK1. One of three synthetic lethal candidates of a synthetic lethal screen with a null allele of MFT1 is shown. Synthetic lethal candidate 20 was transformed with plasmids encoding CTK1, MFT1, SUB2, or an empty plasmid, restreaked onto plates containing 5′-fluoroorotic acid (5′-FOA), and grown for 5 days at 30°C. No growth indicates synthetic lethality. (B) Gbp2 associates with Ctk1 in vivo. TAP-tagged Ctk1 was purified from S. cerevisiae. Copurifying proteins were separated by SDS/PAGE, stained with Coomassie, and identified by mass spectrometry. TAP-tagged Ctk1 is marked by an asterisk. Identified proteins are indicated by numbers and correspond as follows: 1, Kem1; 2, major coat protein, virus L-A; 3, Imd3; 4, Gbp2; 5, Tef2; 6, Ctk2; and 7, Ctk3. Bands indicated by a filled triangle could not be identified unambiguously. Common contaminants are marked by filled squares (heat-shock proteins and ribosomal proteins). (C) TAP-tagged Sub2, Hrp1, Gbp2, or Hrb1 was purified from strains containing Ctk1 tagged with HA. A strain with HA-tagged Ctk1 only served as negative control (wild type). Copurification of Ctk1 was assessed by Western blotting using anti-HA antibodies. (Upper) The SDS/polyacrylamide gel of the EGTA eluate of the TAP purification stained with Coomassie blue. (Lower) The Western blot with anti-HA. The leftmost lanes of A and B show a protein standard.