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. 2010 Mar 15:11:19.
doi: 10.1186/1471-2121-11-19.

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

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A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

Bernardo Cuenca-Bono et al. BMC Cell Biol. .

Abstract

Background: Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts.

Results: In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'-->3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association.

Conclusions: In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

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Figures

Figure 1
Figure 1
Network showing Sus1 interactions. (A) All known physical or genetic SUS1 interactions were downloaded from the BioGRID database [18]. Osprey software was used to obtain the graphical representation of the Sus1 network by gene ontology and a complete legend of colour settings can be found in [26]. (B) Network visualization of SGA genetic interactions involving genes that participate in mRNA processing. Positive SGA interactions are coloured in green, while negative SGA interactions are in red. A complete legend of colour setting and all information can be found in DRYGIN [20].
Figure 2
Figure 2
SUS1 Interacts Genetically with Genes Encoding Components of the mRNA decay machinery. (A) Synthetic lethality of sus1Δ with lsm1Δ and pat1Δ. Double mutants containing a pRS316-SUS1 plasmid, were transformed with an empty vector (pRS313) or the same plasmid bearing a wild-type version of SUS1 (pRS313-SUS1). Transformants were streaked onto 5-fluoroorotic acid (FOA) containing plates, which were incubated at 30°C for 3 days. No growth indicates synthetic lethality. (B) Synthetic sick phenotype of sus1Δ with lsm6Δ or dhh1Δ. Wild-type (wt), single and double mutants were transformed with an empty vector (pRS316). The double mutants were also transformed with a pRS316-SUS1 (pSUS1) in order to complement the phenotype. Cells were diluted in 10-1 steps, and equivalent amounts of cells were spotted on SC-URA plates.
Figure 3
Figure 3
Sus1 co-localises with P-bodies. (A) Sus1 partially localises at P-bodies in stationary phase. Wild-type (wt) cells co-transformed with a plasmid containing the cDNA of SUS1 (pGFP-SUS1cDNA) and a plasmid containing Dcp2 (pDcp2-RFP) were observed in stationary phase. Partial co-localisation indicates localisation of Sus1 in P-bodies. Pictures were obtained by fluorescence microscope. (B) Sus1 is present at P-bodies independently of Lsm1 or Pat1. Cells expressing SUS1cDNA and Dcp2 in the different mutants were observed in stationary phase. Pictures were taken with a camera mounted onto a fluorescence microscope. (C) SUS1 is not required for P-bodies or stress granules formation. Cells expressing Dcp2-RFP or Pab1-GFP were transformed in sus1Δ and observed in stationary phase or after heat shock respectively. Images were generated using a fluorescence microscope.
Figure 4
Figure 4
Sus1 interacts with Dhh1 and co-localise with Pab1. (A) Sus1 physically interacts with Dhh1. Sus1 co-purification with Dhh1 was revealed by western-blot of Sus1-TAP calmoduline eluates purificied from wild-type (wt), and dhh1Δ cells using anti-Dhh1 antibodies (left panel). Prior to purification, Sus1 and Dhh1 presences were confirmed by western blot analysis of whole cell extracts (WCE) (right panel). Lower panel shows the enriched calmoduline eluates from Sus1-TAP (wt), and Sus1-TAPdhh1Δ purifications analysed by SDS 4-12% gradient polyacrylamide gel electrophoresis stained with Coomassie (B) MudPIT analysis of Sus1-TAP. List of proteins co-purified with Sus1 identified by MudPIT. PBs (P-bodies); SGs (stress granules); ND (Not Determined) (C) Sus1 is present at cytoplasmic Pab1-containing granules. Sus1 co-localises with Pab1 in stationary phase.
Figure 5
Figure 5
Absence of the decay mutants partially enhances Sus1 binding to TREX2 and Mex67. Sus1 association to TREX2 is enhanced in pat1Δ and lsm1Δ. Sus1-TAP was affinity-purified from wild-type (wt), pat1Δ and lsm1Δ strains. The enriched calmoduline eluates from all purifications were analysed by SDS 4-12% gradient polyacrylamide gel electrophoresis and proteins stained with Coomassie. Sus1, Tra1, Spt7 and Sac3 bands were verified by mass spectrometry. (B) Sus1 association to Mex67 is increased in the absence of PAT1 or LSM1. Enrichment of Mex67 in the calmoduline eluates from the respectives deletion strains is demonstrated by western blotting using anti-Mex67 antibodies, whereas no differences were observed for Taf6. (C) Sac3, Mex67 and Sus1 expression levels were confirmed by detecting similar loading from inputs by immunoblot.

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