The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

Abstract

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rsslp encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rsslp partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37°C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37°C, the mutant Rat7-lp/Nupl59-lp is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rsslp did not result in retention of the mutant Rat7-lp/Nupl59-lp in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rsslp by placing the RSS1 open reading frame (ORF) under control of the GALl promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rsslp are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rsslp overexpression suppresses the growth defect of rat7-1 cells at 37°C by acting to maintain mRNA export.

Nuclear Export of the Yeast mRNA-binding Protein Nab2 Is Linked to a Direct Interaction with Gfd1 and to Gle1 Function

“…This is based on Mex67/Tap/NXF1 nucleocytoplasmic shuttling and essential interactions with both the RNA-bound Yra1 and nucleoporins. The studies in this paper, combined with previous work, have now linked Gle1 both with an RNA-bound protein (Nab2) and nucleoporins (36,40,46,47,48,58) and have shown Gle1 shuttling or having access to both NPC faces (2,49). Thus, Gle1 is also positioned to play an active role in the translocation mechanism.…”

“…In budding yeast, Gle1 localizes to NPCs at steady state, and gle1 mutants rapidly accumulate poly(A) ϩ RNA in the nucleus (36). GLE1 was initially identified in budding yeast by a synthetic lethal screen with a nup100⌬ mutant (36) and as a multicopy suppressor of a nup159/rat7 mutant (46). Genetic and physical interactions have also been shown between Gle1 and Nup42/Rip1 (36,47,48).…”

Synthetic Genetic Array Analysis in Saccharomyces cerevisiae Provides Evidence for an Interaction Between RAT8/DBP5 and Genes Encoding P-Body Components

“…SGA analysis of the rat8-2 allele: Previous work from our laboratory and many others has demonstrated the value of synthetic lethal analyses for understanding the roles that specific Saccharomyces cerevisiae gene products play in mRNA export as well as identifying novel interactions between mRNA export factors and proteins that function in other processes (Del Priore et al 1996;Murphy et al 1996;Strasser and Hurt 2001;Strasser et al 2002;Gross et al 2007). One focus of our work is the DEAD-box protein and mRNA export factor Rat8p/ Dbp5p.…”

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

“…Immunoelectron microscopy studies show that S. cerevisiae Gle1 is predominantly localized at the cytoplasmic side of the NPC, where it interacts with the cytoplasmic NPC protein Nup42 (16,20,21). Additionally, in S. cerevisiae, Gle1 has functional and physical interactions with several other mRNA export factors and NPC components including Dbp5, Nup159, Nup100, and Gfd1 (16,17,20,[22][23][24]. Deficient Gle1 function results in nuclear mRNA accumulation in a variety of organisms (16,17,19,25), underscoring the conserved role of Gle1 in mRNA export.…”

“…Additionally, in S. cerevisiae, Gle1 has functional and physical interactions with several other mRNA export factors and NPC components including Dbp5, Nup159, Nup100, and Gfd1 (16,17,20,[22][23][24]. Deficient Gle1 function results in nuclear mRNA accumulation in a variety of organisms (16,17,19,25), underscoring the conserved role of Gle1 in mRNA export. Sequence analysis reveals a highly conserved C-terminal domain and a more divergent N-terminal domain (18).…”