This week we profile a recent publication in Nucleic Acids Research from
Dr. Vivien Measday (middle) and Savrina Manhas (second from right) at the UBC Wine Research Centre .
Can you provide a brief overview of your lab’s current research focus?
Our lab uses the budding yeast Saccharomyces cerevisiae (S. cerevisiae) as a model organism to study retroviral replication. S. cerevisiae contains endogenous retroviruses called retrotransposons that replicate via an RNA intermediate and insert new copies back into the genome using the retrotransposon-transcribed enzymes integrase, reverse transcriptase and RNAase H. Yeast retrotransposons are evolutionary ancestors of retroviruses such as human immunodeficiency virus type 1 (HIV-1) and thus share similar gene products and replication cycles to retroviruses. We study the Ty1 retrotransposon which is the most abundant S. cerevisiae retrotransposon. We are interested in discovering host cellular factors that Ty1 requires for genomic integration site selection. The Ty1 element, which is ~6kb in length, inserts into the genome upstream of genes transcribed by RNA polymerase III (RNA Pol III), such as tRNA genes. The rationale behind this targeting mechanism is that tRNA genes are present in multiple copies and have their promoter in the gene coding region. Therefore, targeting the Ty1 element upstream does not impact yeast cell fitness. Previously, our lab and another group discovered that Ty1 integrase, which is required for insertion of Ty1 complementary DNA (cDNA) into the genome, interacts with RNA Pol III subunits and this interaction targets Ty1 elements upstream of Pol III-transcribed genes [1, 2]. However, we felt that the Ty1 targeting mechanism was more complex because tRNA genes can localize to the nuclear pore [3]. As well, Ty1 host factor screens had identified nuclear pore complex (NPC) proteins as important for Ty1 replication [4-6]. The nuclear envelope, which separates the cytoplasm and nucleus, is studded with NPCs that transport macromolecules from the cytoplasm to the nucleus and vice versa. The NPC has transport-independent roles that impact genome organization and transcription. For example, actively transcribed Pol II and Pol III genes can be tethered to the NPC, possibly to enable rapid export of messenger RNA (mRNA) transcripts to the cytoplasm.
What is the significance of the findings in this publication?
The yeast NPC complex, which is highly conserved, is composed of approximately 30 proteins that are arranged in multiple complexes to form a channel that connects the cytoplasm to the nucleus. In this publication, we tested a panel of NPC mutants for defects in Ty1 integration and found that multiple components of the NPC are required for Ty1 replication. Importantly, none of the NPC mutants were required to import Ty1 integrase into the nucleus which meant that another aspect of Ty1 replication was deficient. We tested all aspects of the Ty1 replication cycle including Ty1 transcription, Ty1 mRNA export and translation and Ty1 cDNA production. Some of the NPC mutants had decreased Ty1 translation or cDNA production and we decided to focus on the Nups that were localized on the nuclear side of the NPC which were more likely to interact with chromatin. The nuclear Nups form what is called the nuclear basket and is composed of Nup1, Nup2, Nup60, Mlp1 and Mlp2. If Nup60 is removed, the entire nuclear basket falls off the NPC. We found that removing Nup60 caused a decrease in Ty1 mRNA levels and expression of the Ty1 viral coat protein called Gag. This data was the first indication that the NPC might have a role in Ty1 transcription. However, we were interested in Ty1 targeting and there was an elegant paper that had identified amphipathic helices in both Nup60 and Nup1 and found that the helices were important for tethering the nuclear pore to the nuclear envelope [7]. Dr. Köhler kindly sent us Nup1 and Nup60 mutants that had the helical regions removed but the remainder of the protein intact. This allowed us to identify Nup60 and Nup1 mutants that had wild type levels of Ty1 transcription, but a change in the pattern of Ty1 insertion which was the first indication that the nuclear basket could impact where Ty1 elements insert into the genome. We also found that Nup1 and Nup2 mutants had significantly reduced Ty1 element integration upstream of tRNA genes but Ty1 mobility (the ability of the Ty1 element to insert into the genome) was normal. We found that Ty1 mobility is normal in these mutants because instead of inserting upstream of tRNA genes, the Ty1 element re-targets into sub-telomere regions. The mistargeting of the Ty1 elements to the sub-telomere suggests that nuclear basket Nups are required directly or indirectly, perhaps as global architects or regulators of chromatin organization to orchestrate Ty1 targeting upstream of Pol III-transcribed genes. We would like to acknowledge all the yeast nuclear pore labs sent us their mutant strains and the success of our paper can really be attributed to the generosity of the yeast scientific community that is willing to share their reagents.
What are the next steps for this research?
As stated above, the yeast community was very generous in sending us their mutant strains that we have many avenues for future research. For example, we also received Nup6O sumoylation and ubiquitylation mutants from Dr. Dargemont’s lab and found that the Nup60 SUMO mutant had a more significant reduction in Ty1 insertion upstream of Pol III-transcribed genes than the Nup60 ubiquitylation mutant [8]. This is interesting because the function of Nup60 sumoylation is not known. We also tested a variety of SUMO ligases and found that the Mms21 ligase had a similar reduction in Ty1 insertion as the Nup60 SUMO mutant. Since Mms21 is part of the Smc5/6 complex that functions in DNA repair and genome integrity it will be interesting to determine if Smc5/6 has a role in Ty1 replication. We are also interested in understanding how Ty1 gets redirected to the subtelomeric regions in the absence of nuclear basket proteins. One possibility is that another nuclear pore protein is involved such as Nup170 which interacts with subtelomeric chromatin [9]. Finally, a major goal of the lab is to perform genome-wide Ty1 mapping to identify all new genomic locations where Ty1 is targeted to in the absence of NPC proteins.
This research was funded by:
Canadian Institutes of Health Research (CIHR) Operating Grant HOP-131559
References
- Bridier-Nahmias A, Tchalikian-Cosson A, Baller JA, Menouni R, Fayol H, Flores A, Saib A, Werner M, Voytas DF, Lesage P. Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration. Science 2015; 348(6234):585-588.
- Cheung S, Ma L, Chan PH, Hu HL, Mayor T, Chen HT, Measday V. Ty1 Integrase Interacts with RNA Polymerase III-specific Subcomplexes to Promote Insertion of Ty1 Elements Upstream of Polymerase (Pol) III-transcribed Genes. J Biol Chem 2016; 291(12):6396-6411.
- Chen M, Gartenberg MR. Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast. Genes Dev 2014; 28(9):959-970.
- Dakshinamurthy A, Nyswaner KM, Farabaugh PJ, Garfinkel DJ. BUD22 affects Ty1 retrotransposition and ribosome biogenesis in Saccharomyces cerevisiae. Genetics 2010; 185(4):1193-1205.
- Griffith JL, Coleman LE, Raymond AS, Goodson SG, Pittard WS, Tsui C, Devine SE. Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae. Genetics 2003; 164(3):867-879.
- Risler JK, Kenny AE, Palumbo RJ, Gamache ER, Curcio MJ. Host co-factors of the retrovirus-like transposon Ty1. Mob DNA 2012; 3(1):12.
- Meszaros N, Cibulka J, Mendiburo MJ, Romanauska A, Schneider M, Kohler A. Nuclear pore basket proteins are tethered to the nuclear envelope and can regulate membrane curvature. Dev Cell 2015; 33(3):285-298.
- Nino CA, Guet D, Gay A, Brutus S, Jourquin F, Mendiratta S, Salamero J, Geli V, Dargemont C. Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket. J Cell Biol 2016; 212(2):167-180.
- Van de Vosse DW, Wan Y, Lapetina DL, Chen WM, Chiang JH, Aitchison JD, Wozniak RW. A role for the nucleoporin Nup170p in chromatin structure and gene silencing. Cell 2013; 152(5):969-983.
