Arlen W. Johnson
| Title: | Assistant Professor |  Video - PC Mac |
| Education: | Ph.D.: 1989, Harvard University; B.A.: 1982, University of California-Santa Cruz |
|
| Postdoc.: | Harvard Medical School | |
| Research: | mRNA degradation in yeast | |
| Office: | NMS 1.126 | |
| Phone: | (512) 475-6350 | |
| E-mail: | Arlen@mail.utexas.edu | |
| Postal Address: | Molecular Genetics & Microbiology, A5000, The University of Texas at Austin, Austin, Texas 78712-1095 |
|
| Courses taught: | ||
| Laboratory home page: http://www.bio.utexas.edu/faculty/ajohnson/index.htm | ||
| Work in my lab is focused on two major topics 1) mRNA degradation as a mechanism of translational control and 2) biogenesis of the ribosomal subunits. We use the budding yeast Saccharomyces cerevisiae as a model organism to take advantage of multiple experimental approaches including genetics, genomics and proteomics, biochemistry and cell biology. Transcription is often synonymous with gene regulation. However, many post-transcriptional processes also contribute to gene regulation. Of particular interest to us is mRNA degradation and the interplay between mRNA turnover and translation. Our work initially focused around the function of Xrn1p, a 5'-exoribonuclease of yeast that is responsible for much of the cytoplasmic RNA turnover. A genetic screen based on XRN1 identified a number of different genes that genetically interact with XRN1. These genes fell into three classes. The first class is the superkiller genes, known previously to repress translation of an endogenous RNA virus in yeast and required for a 3'-degradation pathway for mRNA. We have recently shown that Ski2p, a putative RNA helicase, forms a heterotrimeric complex with Ski3p and Ski8p. Our current work is focused on understanding the function of this complex in vivo, identifying additional cellular proteins that interact with this complex and in vitro characterization of the purified complex. A second class of genes that we showed genetically interacted with XRN1 included CDC33, TIF4361 and CEG1, which encode eIF4E, eIF4G and the enzyme responsible for capping mRNAs, respectively. Since mutations in these genes can result in enhanced rates of mRNA degradation, it was surprising that such mutations should be lethal when combined with a deletion of XRN1, which stabilizes mRNAs. We proposed that stabilization of decapped mRNAs by deletion of XRN1 enhances the requirement for cap-dependent translation in vivo. The third class of mutants identified from our screen affected ribosomal subunit biogenesis and was represented by a single allele in NMD3. NMD3 is required for a late step in the production of the large (60S) ribosomal subunit. We have recently shown that Nmd3p contains nuclear import and export sequences and shuttles. In the nucleus Nmd3p binds to nascent 60S subunits and provides the export signal for these subunits to leave the nucleus. Thus, Nmd3p is an adapter protein for 60S subunit export. We have also shown that the receptor for Nmd3p and 60S export is Crm1p/Xpo1p, the receptor for leucine-rich nuclear export signals and well known for its role in human cells mediating the export of HIV rev protein. This is the first identification of the specific export factors for ribosomal subunit export in eukaryotic cells.
Interestingly, Nmd3p is conserved from Archaebacteria to humans and the human ortholog can replace Nmd3p in yeast. The presence of Nmd3p-like proteins in Archaebacteria suggests that Nmd3p has an additional function in subunit biogenesis or translation that predates the evolution of the nucleus. We are pursuing several important questions about Nmd3p and 60S export: 1) What is the role of Nmd3p that is conserved in Archaebacteria? 2) How does Nmd3p interact with the 60S subunit and how is this interaction regulated? 3) Using techniques that we have developed to immunopurify Nmd3p-60S complexes, what is the composition (RNA and protein) of the free 60S complex before and after transport from the nucleus?
|
Selected Publications
2005 |
Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p.
Hedges J, West M, Johnson AW.
EMBO J. 2005 Feb 9;24(3):567-79. Epub 2005 Jan 20.
2003 |
The Putative GTPases Nog1p and Lsg1p Are Required for 60S Ribosomal Subunit Biogenesis and Are Localized to the Nucleus and Cytoplasm, Respectively.
Kallstrom, G., Hedges, J. and Johnson AW.
Mol Cell Biol. 2003 Jun;23(12):4344-55.
Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates.
Trotta CR, Lund E, Kahan L, Johnson AW, Dahlberg JE.
EMBO J. 2003 Jun 2;22(11):2841-51.
2002 |
Nuclear export of ribosomal subunits.
Johnson AW, Lund E, Dahlberg J.
Trends Biochem Sci. 2002 Nov;27(11):580-5.
2001 |
Nmd3p Is a Crm1p-dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit
Ho JH, Kallstrom G, Johnson AW.
J Cell Biol 2000 Nov 27;151(5):1057-1066.
Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p
Ho JH, G. Kallstrom, and A.W. Johnson.
RNA 2000 Nov;6(11):1625-34.
2000 |
Brown JT, Yang X, and A.W. Johnson.
Genetics 155 (2000): 31-42.
ABSTRACT: Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts. A screen for mutations synthetic lethal with xrn1Delta identified a mutation in CDC33, encoding eIF4E. This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G. Synthetic lethality between xrn1 and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p. High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant. Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not. Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs. Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1Delta. Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex.The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo
Brown JT, Bai X, Johnson AW.
RNA 6 (2000): 449-457.
ABSTRACT: The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p.
Xue Y, Bai X, Lee I, Kallstrom G, Ho J, J. Brown, A. Stevens, A.W. Johnson.
Molecular and Cellular Biology 20 (2000): 4006-4015.
ABSTRACT: The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5. 8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.
1999 |
Ho, J. and A.W. Johnson.
Molecular and Cellular Biology 19 (1999): 2389-2399.
ABSTRACT: A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.
1998 |
Page, A.M., K. K. Davis, C. Molineux, R. D. Kolodner and A. W. Johnson.
Nucleic Acids Research 26 (1998): 3707-3716.
ABSTRACT: Exoribonuclease I from yeast is a 175 kDa protein that is responsible for the majority of cytoplasmic mRNA degradation. Alignment of the Xrn1p sequence with homologs from yeast as well as from higher eukaryotes suggests that the protein is composed of several domains: two acidic N-terminal domains which likely contain the exonuclease, a basic middle domainand a basic C-terminal domain. Deletion analysisdemonstrated that the C-terminus is dispensable for most in vivo and in vitro functions but confers a dominant negative growth inhibition when expressed at high levels. This growth inhibition is not due to the exonuclease function of the protein. To identify specific residues responsible for in vivo function, a screen was carried out for non-complementing missense mutations. Fourteen single point mutations were identified that altered highly conserved amino acids within the first N-terminal domain of Xrn1p. All of the mutations reduced exonuclease activity measured in vivo and in vitro using affinity-purified proteins. The mutants fell into two phenotypic classes, those that reduced or abolished exonuclease activity without qualitatively changing the products of RNA degradation and those that gave rise to novel degradation intermediates on certain RNAs.
1997 |
Johnson, A.W.
Molecular and Cellular Biology 17 (1997): 6122-6130.
ABSTRACT: XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.
1995 |
Johnson, A.W., and R. Kolodner.
Molecular and Cellular Biology 15 (1995): 2719-2727.
ABSTRACT: Strand exchange protein 1 (Sep1) (also referred to as exoribonuclease I [Xrn1]) from Saccharomyces cerevisiae has been implicated in DNA recombination, RNA turnover, karyogamy, and G4 DNA pairing among other disparate cellular processes. Using a genetic approach to study the role of SEP1/XRN1 in mitotic yeast cells, we identified mutations in the genes superkiller 2 (SKI2) and superkiller 3 (SKI3) as synthetically lethal with an sep1 null mutation. The SKI genes are thought to comprise an intracellular antiviral system controlling the expression of killer toxin from double-stranded RNA virus found in many yeast strains. However, the lethality of sep1 ski2 and sep1 ski3 mutants was independent of the L-A and M viruses, suggesting that the SKI genes act in a general cellular process in addition to virus control. We propose that Sep1/Xrn1 and Ski2 both act to block translation on transcripts targeted for degradation. Using a temperature-sensitive allele of SEP1/XRN1, we show that double mutants display a synthetic cell cycle arrest in late G1 at Start.
(a new browser window will open)
