Gbp2 interacts with THO/TREX through a novel type of RRM domain

Abstract

Metazoan SR and SR-like proteins are important regulatory factors in RNA splicing, export, translation and RNA decay. We determined the NMR structures and nucleic acid interaction modes of Gbp2 and Hrb1, two paralogous budding yeast proteins with similarities to mammalian SR proteins. Gbp2 RRM1 and RRM2 recognise preferentially RNAs containing the core motif GGUG. Sequence selectivity resides in a non-canonical interface in RRM2 that is highly related to the SRSF1 pseudoRRM. The atypical Gbp2/Hrb1 C-terminal RRM domains (RRM3) do not interact with RNA/DNA, likely because of their novel N-terminal extensions that block the canonical RNA binding interface. Instead, we discovered that RRM3 is crucial for interaction with the THO/TREX complex and identified key residues essential for this interaction. Moreover, Gbp2 interacts genetically with Tho2 as the double deletion shows a synthetic phenotype and preventing Gbp2 interaction with the THO/TREX complex partly supresses gene expression defect associated with inactivation of the latter complex. These findings provide structural and functional insights into the contribution of SR-like proteins in the post-transcriptional control of gene expression.

hLARP7 C-terminal domain contains an xRRM that binds the 3′ hairpin of 7SK RNA

“…pseudoRRMs (73) and occluded RRMs (oRRMs) (74). Some atypical RRMs have an α-helix on the β-sheet, but this helix is not used for RNA binding: it either binds protein (75,76) and the RRM is not involved in RNA recognition (77–80), or moves out of the way for RNA binding (81,82). Although other atypical RRMs contain some unusual features in common with the xRRM, the combination of helix α3-α3x and conserved RNP3 sequence that together form a unique RNA–protein binding interface sets the xRRM apart.…”

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

“…Plasmids used in this work are summarised in Supplementary Table S1 . Nrd1 sequences were amplified (KOD polymerase; Novagen) from Saccharomyces cerevisiae genomic DNA and cloned in a pET28-modified vector containing an N-terminal fusion cassette (thioredoxin A+6xHis+TEV sequence) (( 27 ) for details). Mutants were obtained with a QuikChange Lightning Kit (Agilent genomics), both in the pET28 and pRS415 plasmid backgrounds using specific DNA oligos (IDT and Macrogen).…”

“…Upon reaching OD 600nm = 0.6–0.8, cells were transferred to 12°C and induced with 0.5 μM IPTG (Sigma-Aldrich) during 72–96 h. We noticed that expression at higher temperatures result in production of misfolded protein. Uniformly ( 15 N and/or 13 C) or selectively unlabeled samples (see specific details in Supplementary Figure S1 ) were produced in a similar way in chemically defined media (( 27 ) for details). Selenomethionine substituted Nrd1 301–489 was expressed similarly, just using minima media supplemented with a mix of inhibition aminoacids and Selenomethionine.…”

“…Measurements were performed in a BMG Polarstar Galaxy plate reader essentially as described in ( 27 ). The temperature was 26°C, the concentration of the fluorescein labeled oligonucleotides (IDT) was 40 nM, and the buffer was 20 mM Tris–HCl, 150 mM NaCl, 1 mM DTT pH 8.0.…”

The role of TREX in gene expression and disease

“…Since NXF1 requires both an adaptor and a co-adaptor for optimal RNA binding, this raises the question of whether SR proteins function independently to recruit NXF1 to mRNAs, given they would only provide the adaptor function. Evidence from yeast suggests that SR proteins may function in collaboration with TREX during mRNA export [133–135], and this may be to ensure that a co-adaptor is available at such sites for optimal NXF1 RNA binding.…”