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. 2013 Oct;19(10):1363-71.
doi: 10.1261/rna.040048.113. Epub 2013 Aug 20.

Mutational Analysis of the Yeast RNA Helicase Sub2p Reveals Conserved Domains Required for Growth, mRNA Export, and Genomic Stability

Free PMC article

Mutational Analysis of the Yeast RNA Helicase Sub2p Reveals Conserved Domains Required for Growth, mRNA Export, and Genomic Stability

Cyril Saguez et al. RNA. .
Free PMC article

Abstract

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.

Keywords: RNA; RNA export; RNA helicase; genome stability.

Figures

FIGURE 1.
FIGURE 1.
Yeast Sub2p contains a conserved N-terminal motif (NTM) required for normal growth. (A) WebLogo showing conservation of the Sub2p NTM as retrieved from 186 nonredundant protein sequences (Crooks et al. 2004). Numbering denotes the position of amino acids in S. cerevisiae Sub2p. The y-axis indicates the relative frequency of each amino acid. (BD) Tenfold serial dilutions of indicated S. cerevisiae sub2 NTM mutant strains plated for 3 d on 5-FOA plates at 25°C. Lines and rectangles denoting “NTM,” “linker,” “random sequence,” “core helicase domain,” and deleted residues are explained below image D.
FIGURE 2.
FIGURE 2.
Mutation of the Sub2p NTM causes nuclear accumulation of poly(A)+ RNA. (A) Poly(A)+ RNA-FISH analysis of the indicated yeast strains grown at 25°C or heat-induced at 37°C for 15 min before fixation and processing. A Cy3-labeled and LNA-spiked dT20 probe was used for poly(A)+ targeting, and DAPI staining visualized the chromatin-rich part of the nuclei. (B) Western blotting analysis of proteins expressed in A. Cells were harvested after 0 or 3 h of growth at 37°C as indicated. Membranes were sequentially probed with α-Sub2p and α-Nop1p (loading control) antibodies. (C) Recruitment of HA-tagged Sub2p wt or indicated NTM mutants to the PMA1 gene at 25°C (top) or 37°C 15′ (bottom) analyzed by ChIP with amplicons directed toward the 5′ end (5′), middle (M), or 3′end (3′) of the gene. HA-tagged fusion proteins were expressed in a Δsub2 background and α-HA ChIP levels were normalized to Rpb3p ChIP values obtained from the same extracts and to Sub2p-HA wt levels from the same experiment. Average signals and standard deviations reflect ChIP results from three different chromatin extracts.
FIGURE 3.
FIGURE 3.
Mutation of the NTM does not affect Sub2p helicase activity. (A) Schematic representation of mutations in the helicase core of Sub2p analyzed in this study. Location of the characteristic helicase motifs in DEAD-box proteins (red, motifs involved in ATP binding and hydrolysis; blue, motifs involved in RNA binding; green, motifs involved primarily in coupling between ATP and RNA binding). Boxes show the DEAD-box family consensus in motifs Ia, Ic, and II (Fairman-Williams et al. 2010); corresponding sequences of Sub2p are given underneath. The label for E216 is omitted for clarity. (B) In vitro RNA-binding gel-shift assay using a single-stranded 5′-radiolabeled 13-mer RNA. The migration of free and protein-bound RNA is shown. Sub2 was present at a concentration of 2 µM and the RNA at 0.6 nM. (C) ATPase activity measured by a modified Baginski-type assay (Cariani et al. 2004) using wt Sub2p or mutant variants as indicated and poly(U). The assay measures the relative production of Pi in nmol during a fixed time interval. (D) RNA-unwinding assay using a 13-bp duplex substrate with 12-bp overhang and wt or mutant Sub2p protein without (−) or with (+) ATP/Mg, as indicated. The positions of duplex substrate and unwound product are indicated at the left, where the stars show the position of the radiolabel. (E) RNA:DNA hybrid unwinding assay as in D but using a duplex of a 13-nt DNA annealed to a 38-nt RNA. (F) Tenfold serial dilution of yeast Δsub2 cells complemented by plasmids expressing wild type (SUB2), mutant Sub2p variants, or an empty vector control as indicated. Cells were plated on 5-FOA plates for 3 days at 25°C.
FIGURE 4.
FIGURE 4.
Sub2p helicase mutants display dominant negative growth and recombination phenotypes. (A) Tenfold serial dilutions of cells plated for 3 d at 25°C or 37°C and transformed with plasmids expressing either wt or mutant Sub2p as indicated. (B) Cells carrying the integrated leu2k–ADE–URA–leu2k repeat system (bottom) were used as vehicles to examine the impact of ectopic expression of Sub2p mutants on genomic stability. The frequency of recombination was measured as previously described (Prado and Aguilera 1995) with the hyper-recombinant Δhpr1 strain as a positive control. (C) Recruitment as analyzed by ChIP of HA-tagged (top) or total (bottom) Sub2p to PMA1 (left) or HSP104 (right) genes after a 37°C heat induction for 15 min. Cells were transfected with centromeric plasmids expressing HA-tagged wt SUB2 or its mutant variants, as indicated. An α-HA antibody was used to specifically precipitate exogenous HA-tagged proteins, and α-Sub2p antibody was used to precipitate total Sub2p (exogenous and endogenous proteins). ChIP levels as measured to the 5′, middle, or 3′ regions of the genes were normalized to Rpb3p values and to levels of wt Sub2p cells from the same samples. Average signals and standard deviations reflect ChIP results from three different chromatin extracts.

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