Sem1

Abstract

The evolutionary conserved protein Sem1/Dss1 is a bona fide subunit of the 19S regulatory particle (RP) of the proteasome and in mammalian cells stabilizes the tumor suppressor protein BRCA2. A recent study from our laboratory has revealed an unexpected non-proteasomal role of Sem1 in mRNA export. We found that Sem1 is the unique subunit of the RP that is required for proper nuclear export of mRNAs, transcription elongation and preventing transcription-associated hyper-recombination. Biochemical analyses revealed that Sem1, independent of the RP, coenriched with the TREX-2 complex that is required for tethering a subset of genes to the nuclear periphery, transcription-coupled mRNA export and preventing transcription-associated genome instability. Strikingly, we found that Sem1 coenriched with a third multi-subunit protein complex namely the COP9 signalosome, which is involved in deneddylation. We propose that Sem1 is a versatile protein that regulates the functional integrity of multiple protein complexes involved in diverse biological pathways.

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Introduction

In all eukaryotes, RNA polymerase II (RNAPII) initiates the first step in gene expression in the nucleus, leading to production of mRNAs. Emerging nascent pre-mRNAs are modified, processed and matured into mRNAs by several protein complexes that are loaded onto transcription sites via their interaction with the C-terminal domain of RNAPII.1–3 Processing and maturation events of the pre-mRNAs include the addition of a 5′-cap, splicing, 3′ cleavage and subsequent poly-adenylation. There is growing consensus that transcription, pre-mRNA processing and the export of mature mRNA to the cytoplasm are coupled.2–5

In budding yeast and mammalian cells the THO/transcription export (TREX) complex was shown to connect biogenesis of mRNPs with their nuclear export.4 THO/TREX is thought to be recruited to the elongating RNAPII via the THO subunits (Hpr1, Tho2, Mft1 and Thp2), which function in transcription and packaging of mRNA protein complexes (mRNPs). The additional TREX factors Sub2 and Yra1 are involved in recruiting the hetero-dimeric export receptor Mex67-Mtr2 to nascent mRNPs (Fig. 1A), thereby coupling mRNP biogenesis to nuclear export (reviewed in refs. 2, 4 and 6). A genetic screen with Yra1 identified an additional transcription-mRNA export complex, called TREX-2 consisting of Sac3, Thp1, Sus1 and Cdc31 (Fig. 1B). TREX-2 potentially coordinates Spt7-Ada2-Gcn5 acetyltransferase (SAGA)-mediated trans cription of a subset of genes at the nucleoplasmic face of the NPC.7 A central component of TREX-2 is Sac3, a multi-domain protein that serves as a binding platform for other members of the complex. The N-terminal and middle domain (N + M) of Sac3 bind Thp1 and Mex67-Mtr2, while the C-terminal domain mediates its NPC targeting8 and recruits the centrin Cdc31 as well as Sus1.9,10 Recent works from several groups have demonstrated a requirement of TREX-2 in “gene gating,” a process involving the dynamic repositioning of a subset of gene loci from the nuclear interior to the nuclear periphery upon their activation.11–15

Sem1 is a Functional Component of the TREX-2 Complex

Sem1 is a small acidic protein that is highly conserved among all eukaryotic species. SEM1 was originally isolated as a multi-copy suppressor of exocyst mutants in budding yeast.16 Mutations in Sem1 lead to several pleiotropic phenotypes such as defects in exocytosis, pseudohyphal growth and defects in the cell cycle.16,17 Genetic screens and proteomic approaches identified Sem1 as a component of the lid sub-complex of the regulatory particle (RP) of the proteasome in both budding yeast and humans.18–21 Loss of Sem1 impairs the functional integrity of the RP.18 Consistent with this, sem1Δ mutants show impaired ubiquitin-dependent protein degradation and accumulate polyubiquitinated proteins.21 The deletion of Sem1/Dss1 in fission yeast was shown to be defective in the nuclear export of mRNAs.22,23 Curiously, in budding and fission yeasts Sem1 was reported to purify only the RP of the proteasome, implicating the ubiquitin- proteoasome pathway in the nuclear export of mRNAs.24,25 Therefore, we sought to shed light on the direct role of the RP, if any, in nuclear export of mRNAs using budding yeast as a model system.

Our recent work has provided several lines of evidence that Sem1 has an additional moonlighting role in mRNA export as part of TREX-226 (Fig. 1B). Like impaired TREX-2 mutants, sem1Δ cells were defective in nuclear export of mRNAs.8 Since Sem1 was identified as a bona fide subunit of the RP we tested whether a functional RP is required for proper mRNA export. However, neither mutants impaired in RP function nor directly inhibiting proteasomal activity by the drug MG132 induced defects in nuclear export of mRNAs. Thus, Sem1 is the unique component of the RP that is required for proper nuclear export of mRNAs. Further, only Sem1, but not other components of the RP, is genetically linked to essential components of the mRNA export pathway (Mex67, Mtr2, Yra1 and Sub2). Finally, the overexpression of Sub2 strongly inhibited growth of sem1Δ, in a manner similar to TREX-2 mutants.27,28 Importantly, growth of other RP mutants was found not to be sensitive to Sub2 overexpression. The sensitivity of sem1Δ to Sub2 overexpression raised the possibility of a direct physical association with TREX-2. To test this, using Thp1 and Sac3 as bait proteins, we directly purified TREX-2 and showed that Sem1 specifically co-enriched in these purifications, independent of the RP. Importantly, the enrichment of Sem1 with TREX-2 was found to be RNA independent. Together these cell-biological, genetic and biochemical data suggest that, like in the 19S RP, Sem1 is a bona fide component of TREX-2. Interestingly, a combination of an unbiased large-scale genetic screen and biochemical approaches towards understanding RNA processing also identified Sem1 as a novel functional component of TREX-2.29

Why was the association of Sem1 with TREX-2 not previously observed? It appears that the addition of the large TAP epitope abolishes its association with TREX-2. Consistent with this possibility Sem1-TAP is non-functional in the mRNA export pathway, as the Sem1-TAP strain showed a defect in mRNA export and, when combined with mex67 and mtr2 mutants was found to be synthetically lethal.

Sem1 Associates with Sac3_GANP and PAM Domain Containing Protein Complexes

Do the RP and TREX-2 contain common structural features that are recognized by Sem1? Indeed, both the RP and TREX-2 contain protein pairs that exhibit Sac3_GANP and PAM domains (Fig. 2).30–32 The Sac3_GANP domain is present in Rpn12 (RP) and Sac3 (TREX-2), whereas Rpn3 (RP) and Thp1 (TREX-2) contain the PAM domains (Fig. 2).30,32 Both the Sac3_GANP and PAM domains are predicted to be alpha-helical and have been proposed to mediate protein-protein interactions.30 Intriguingly, bioinformatic analyses revealed that Csn12, a subunit of the COP9 signalosome (CSN), contains a PAM domain.30 Further, in the Csn12-TAP purification, proteomic approaches identified a Sac3_GANP domain containing protein, Ypr045c.33 We tested whether Sem1 also co-enriched with these components and found that this was indeed the case. Deletions of members of the CSN complex have been shown to accumulate neddylated cullin Cdc53.34 However, we did not observe any alterations in Cdc53 de-neddyltion in sem1Δ, ypr045cΔ and csn12Δ strains. Moreover, we found that both csn12Δ and ypr045cΔ strains are not defective in mRNA export, suggesting that Sem1 has an additional role in the CSN that is independent of its function in the RP and TREX-2. Interestingly, Wilmes et al. have shown that the Csn12-Ypr045c-Sem1 sub-complex associates with the splicing machinery and that the csn12Δ and ypr045c strains were defective in pre-mRNA splicing. Thus, the Csn12-Ypr045c-Sem1 sub-complex is likely to affect a yet undetermined activity of the CSN. We propose that Sem1 specifically associates with at least three distinct protein complexes that contain known Sac3_GANP and PAM domains (Fig. 2).

What could be the Role of Sem1 in the RP, TREX-2 and CSN?

Two scenarios can be envisioned for the formation of Sem1 containing complexes (Fig. 3). In one scenario Sem1 might stabilize interactions between the Sac3_GANP containing subunit and its PAM domain counterpart (Fig. 3A). Another possibility could be that Sem1 stabilizes the PAM domain which then would facilitate a robust interaction with the Sac3_GANP containing subunit or vice versa (Fig. 3B). Consistent with the first possibility, we found that the tethering of Thp1 to the NPC was severely affected in sem1Δ and that the association of Sem1 with TREX-2 required the presence of both Sac3 and Thp1. Whether Sem1 directly “glues” the Sac3_GANP and PAM domains together still remains to be determined. Evidence favoring the second possibility is the finding that Sem1/Dss1 maintains the correct conformation of BRCA2, as BRCA2 is largely insoluble in absence of Dss1.35,36 Structural analyses of Sac3_GANP and PAM domains in complex with Sem1 will provide further insights into how Sem1 might stabilize protein complexes.

Can Sem1 associate with other protein complexes? Here the crystal structure of Sem1/Dss1 in complex with a part of BRCA2 can provide possible insights. Sem1/Dss1 was shown to bind the helix turn helix motif (helical domain) and an oligonucleotide-binding domain (OB) of BRCA2 in an extended conformation. Sem1/Dss1 appears to make contacts with numerous residues on the helical domain and the OB surface that is rich in basic, aromatic and hydrophobic residues.36 The Sac3_GANP and PAM domains contain periodic hydrophobic patches followed by conserved positive residues found typically in α-helical structures.31,32 Interestingly, Sac3_GANP and PAM domains have neither structural nor sequence homology with the region of BRCA2 that interacts with Sem1. In addition to the proteasome and BRCA2, in human cells, Dss1 was shown to associate with the Integrator complex (involved in snRNA processing).37 Interestingly, the Integrator complex appears not to contain an obvious Sac3_GANP and PAM domain protein pair. Hence, it seems possible that Sem1 targets multiple folds/surfaces present in several unrelated protein complexes. Genetic approaches in budding yeast need to be further exploited to reveal additional biological processes that Sem1 might regulate.

A Non-Proteasomal Role for Sem1/Dss1 in Preventing Transcription-Associated Genomic Instability

A large body of work in mammalian cells has implicated mutations in the tumor suppressor protein BRCA2 in genomic instability. BRCA2 functions in the repair of double-strand breaks (DSBs).38–40 Sem1/Dss1 was discovered to be a BRCA2-binding protein17 that specifically interacts with the C-terminal portion of BRCA2.36 Sem1/Dss1 has been shown to stabilize BRCA2 and consistently, its depletion in mammalian cells was shown to induce phenotypes similar to those seen in BRCA2-deficient cells.27,40,41 Although budding yeast lacks an obvious homologue of BRCA2, Sem1, as part of the RP was shown to be important for the repair of DSBs by both homologous recombination and non-homologous end joining pathways.20

In addition to their direct function in mRNA export, both THO/TREX and TREX-2 play an important role in preventing transcription-associated genomic instability.6 THO/TREX and TREX-2 mutants induce co-transcriptional formation of RNA:DNA hybrids (R-loops) between the emerging RNA and the transcribed single stranded DNA (ssDNA).42 R-loops are believed to become obstacles for subsequent elongating RNAPIIs, thus impairing transcription elongation or generating mRNA-RNAPII-DNA tertiary structures that can obstruct replication, leading to genome instability.43 TREX-2 mutants induce transcription- associated hyper-recombination phenotypes that are very similar to those seen in sem1Δ cells. Thus, our work has uncovered a non-proteosomal role of Sem1 in preventing transcription-associated genomic instability by contributing to the functional integrity of TREX-2.

Concluding Remarks

Sem1 was previously identified as a bona fide subunit of the RP and a stabilizer of the tumor suppressor protein BRCA2. Our recent work has identified Sem1 as a component of two other protein complexes TREX-2 and the CSN, which are involved in distinct biological pathways. The precise role of Sem1 in the CSN still remains to be unraveled. It is intriguing that despite their functional diversity the RP, TREX-2 and CSN contain common structural elements: Sac3_GANP and PAM domains a shared functional component: Sem1/Dss1. Determining at the molecular level the common principle by which Sem1 modulates the stability and hence the functioning of these important biological machines remains a future challenge.

Figures and Tables

Figure 1 mRNA export and gene gating in S. cerevisiae. (A) The THO complex is co-transcriptionally recruited and associates with nascent transcripts. Together with Sub2 and Yra1 the trEx complex is formed. Mex67-Mtr2 binds to the mrNPs through adaptor proteins such as Yra1 and facilitates their nuclear export. (B) Interactions between the SAGA transcription initiation complex and the nuclear pore associated TREX-2 complex result in tethering of activated genes to the nuclear periphery.

Figure 2 Association of Sem1 with target complexes. The RP of the proteasome, TREX-2 and the CSN are the three complexes in budding yeast, which contain PAM and Sac3_GANP domains and feature Sem1 as a component.

Figure 3 Models for interaction of Sem1 with Sac3_GANP and PAM domains. Depending on the ability of Sem1 to interact with PAM and Sac3_GANP domains individually, we propose two distinct models: in (a) Sem1 is not able to interact with PAM or Sac3_GANP domains individually and therefore association of Sem1 necessitates an interaction of these domains prior to binding. In model (B), Sem1 can interact with PAM and Sac3_GANP domains individually and thereby bridge these domains.

Acknowledgements

V.G. Panse is supported by grants from the Swiss National Science Foundation and Swiss Federal Institute of Technology.