Undergraduate thesis biochemistry & molecular biology

For more than three years I worked in Deborah Lycan’s molecular biology lab at Lewis & Clark, where I studied ribosome biogenesis in a laboratory variant of bakers yeast. In the spring of 2009 I completed a 40 page research thesis, summarized below. Ultimately I was awarded departmental honors for successfully completing the written thesis, giving a seminar to faculty, students in my program, and a few daring members of the general public (slides above), and orally defending the thesis before the BCMB faculty.

Deborah was an extraordinary mentor, and over the course of several years became a dear friend. Greg Hermann provided critical feedback as my thesis reader, and I also appreciate the support of department chair Janis Lochner, and the rest of BCMB faculty for cultivating my understanding of science during those 4 wonderful years at Lewis & Clark.

Background

The yeast S. cerevisiae can complete its cell cycle in as little as ninety minutes. Reproducing all of the protein that underlies cell structure and function requires the constant activity of over 200,000 ribosomes per cell. Ribosome production reaches rates as high as 33 new ribosomal subunits every second, and is the primary energetic cost for rapidly growing yeast cells and the primary limit on the rate at which cells can divide (Warner 1999).

Ribosome biogenesis is a highly conseved process in eukaryotic organisms, beginning in the nucleolus where three major rRNAs, the 18S, 5.8S, and 25S, are transcribed as a single 35S pre-rRNA precursor. Ribosomal proteins, non-ribosomal proteins, and small nucleolar ribonucleoprotein particles bind to the nascent rRNA, ultimately forming a large 90S pre-ribosome. Cleavage of the 35S rRNA splits the 90s pre-ribosome into a pre- 40S pre-ribosomal subunit and a larger pre-66S subunit. The 40S subunit sheds most of the 30+ processing factors associated with the 90S particle and is exported from the nucleus into the cytoplasm shortly after the 90S cleavage (Schafer et al., 2003).

Small Subunit Export

Nuclear export of macromolecules such as the ribosomal subunits is regulated by export and import receptors called karyopherins. Crm1 is the major export karyopherin in yeast; it binds and exports proteins that have a conserved, leucine-rich nuclear export signal [NES]. Neither ribosomal subunit is thought to contain any NES elements in cis; instead, Crm1 mediates large subunit export by binding an export adapter, Nmd3, which contains an NES. Nmd3 in turn binds the 60S to enable export (Ho et al., 2000). Although small subunit export also depends on Crm1 (Moy and Silver 1999), no adapter analogous to Nmd3 has yet been identified. The Lycan lab has proposed that Ltv1 is a non-essential export adapter for the 40S subunit (Seiser et al., 2006). Ltv1 is coded by a nonessential gene, and it shuttles in and out of the nucleus in a Crm1-dependent manner. Disruption of Ltv1 affects the cellular localization of components of the 40S subunit (Seiser et al. 2006, Loar et al. 2004). If Ltv1 is a Crm1-dependent export adapter, it should bind Crm1 directly and also bind the 40S subunit. Ltv1 has been independently reported to interact with Crm1 in two hybrid assays, and the Lycan lab recently found that deletion of an inhibitory 43aa N terminus of Ltv1 is necessary for the two-hybrid interaction between Ltv1 and Crm1 (unpublished observations). In addition, Several lines of evidence indicate that Ltv1 binds the 40S subunit. Ltv1 co-sediments in sucrose gradients with the 43S/40S subunit (Loar et al., 2004), and co-precipitates with late pre-40S subunits when affinity purified with TAP-tagged Ltv1 or Enp1 (Schafer et al., 2006).

Ribosomal protein Rps3 May Anchor the Adapter Complex

The Lycan lab began investigating the small subunit protein Rps3 as a potential site at which Ltv1 may anchor the Crm1 export complex to the small subunit after confirming a reported two-hybrid interaction between Ltv1 and RpS3 (Ito et al, and unpublished observations). RpS3 is a conserved 40S sub-unit protein with proposed roles in translation initiation (Westermann et al. 1981) and decoding accuracy (Hendrick et al. 2001). A role for Rps3 in 40S export is supported by experiments that show that cells depleted of Rps3 specifically accumulate 20S rRNA in the nucleoplasm, a phenotype associated with a specific defect in export rather than processing (Ferreira-Cerca et. al. 2005).

Two additional proteins that interact with both Rps3 and Ltv1 are of interest because they may also function in 40S export. For example, In vivo, a complex containing Rps3, Ltv1 and 40S, and the biogenesis factor Enp1 can be salt extracted from 43S pre-ribosomes, but not mature cytoplasmic 40S ribosomes (Schafer et al., 2006). Phosphorylation of Rps3 by the kinase Hrr25 also leads to dissociation of the complex in vitro, and Schafer et al. suggest that this phosphorylation may mediate conformational flexibility that enables the small subunit to fit through the nuclear pore (Schafer et al., 2006). In addition to Enp1, the small ankyrin-repeat protein Yar1 is of interest because it physically interacts with Rps3 as well as with Ltv1. Mutants lacking Yar1 have aberrant polysome profiles, with a reduced number of 40S subunits and excess of free 60S subunits. Over expression of RPS3 in the Δyar1 mutants suppresses this defect, suggesting that Yar1, in connection with Rps3, has a non-essential role in 40S biogenesis (Loar et al., 2004).

Generation and Analysis of RPS3 Mutants

To explore Rps3’s role in small ribosomal subunit assembly and function, previous thesis student Kelsey Rogers generated a library of random point mutations in the RPS3 gene, and inserted the mutagenized fragment in a vector that contained Green Fluorescent Protein and the native RPS3 promoter. Rogers screened for mutants using a yeast strain in which the Gal1 promoter has been integrated upstream of the chromosomal RPS3 gene. This means that the endogenous RPS3 gene is expressed when cells grow on galactose and repressed when cells are grown on glucose. The result is that yeast containing a plasmid which expresses non-functional RPS3 should grow on galactose but not glucose, whereas expression of wild type RPS3 from the plasmid should produce viable yeast on either medium. Screening for a glucose-dependent mutant phenotype helps distinguish between mutations in RPS3 (the only gene whose expression is sugar-dependent) and random mutations in the genome that may have occured during transformation of the plasmid into yeast.

In this thesis, I characterized the collection of RPS3 mutants that Kelsey Rogers had generated. I recovered 19 plasmids from yeast, purified the DNA, and transformed them back into yeast to test if the plasmids could reconfer the original slow-growth phenotype. I used a more sensitive assay for growth this time and screened for nonconditional mutants that exhibited growth defects. I used GFP microscopy to test if any of these mutants were specifically defective in 40S export as predicted by our model for 40S export. I determined the sequence of confirmed mutant plasmids and mapped the mutations onto the proposed structure of yeast Rps3. Interestingly, all 6 mutations clustered to the solvent exposed surface of RpS3, as opposed to surfaces likely to interact with either the rRNA or with other small subunit ribosomal proteins. Finally I am testing whether these mutations affect RpS3’s interaction with either Ltv1 or Yar1 by testing whether over expression of either suppresses the slow growth phenotype of any of these Rps3 mutants.

If you just have time for one background reference, check out this recent paper by our lab.

Seiser, R. M., Sundberg, A. E., Wollam, B. J., Zobel-Thropp, P., Baldwin, K., Spector, M. D., and Lycan D. E. (2006). Ltv1 Is Required for Efficient Nuclear Export of the Ribosomal Small Subunit in Saccharomyces cerevisiae. Genetics 174, 679-691.

Complete List of References

De Boulle, K., Verkerk, A.J., Reyniers, E., Vits, L., Hendrickx, J., Van Roy, B., Ban den Bos, F., de Graaff, E., Oostra, B.A. and Willems, P.J. (1993) A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nature Genet., 3, 31-35.


Fatica, A., and Tollervey, D. (2002). Making Ribosomes. Curr. Opin. Cell Biol. 14, 313-318.

Gibson, T.J., Thompson, J.D. and Heringa, J. (1993) KH domains within the FMR1 sequence suggest that fragile X syndrome stems from a defect in RNA metabolism. Trends Biochem. Sci., 18, 331-333.


Grishin, N.V. 2001. KH domain: One motif, two folds. Nucleic Acids Res. 29: 638–643.

Ho, J. H., Kallstrom, G. and Johnson, A.W. (2000). Nmd3 is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151, 1057-1066.


Fatica, A., and Tollervey, D. (2002). Making Ribosomes. Curr. Opin. Cell Biol. 14, 313-318.


Ferreira-Cerca, S., G. Poll, P. Gleizes, H. Tschochner, and P. Milkereit. (2005). Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function. Mol. Cell 20: 263-275.


Ito, T., T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, 2001 A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98: 4569-4574.


Jensen, T. H., Neville, M., Rain, J.C., and Rosbash, M. (2000). Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor Ace2p shows cell cycle-independent nucleocytoplasmic shuttling. Mol. Cell. Biol. 20, 8047-8058.


Johnson, A. W., E. Lund, and J. Dahlberg, 2002 Nuclear export of ribosomal subunits. TIBS 27: 580-585.


Loar, J. W., R. M. Seiser, A. E. Sunderberg, H. J. Sagerson, N. Ilias et al., 2004 Genetic and biochemical interactions among Yar1, Ltv1, and RPS3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae. Genetics 168: 1877-1889.


Moy, T. I., and P. A. Silver, 1999 Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev 13: 2118-2133.


Moy, T. I., and P. A. Silver, 2002 Requirements for the nuclear export of the small ribosomal subunit. J Cell Sci 115: 2985-2995.

Neuber, A., Franke, J., Wittstruck, A., Schlenstedt, G. Sommer, T., Stade, K. (2008). Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast. Mol. Cell. Biol. No. 20, 0270-7306

Oldenburg, K. R., K. T. Vo, S. Michaelis, and C. Paddon. (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25: 451-452.

Passmore, L. A., T. M. Schmeing, D. Maag, D. J. Applefield, M. G. Acker, M. A. Algire, J. R. Lorsch, and V. Ramakrishnan, 2007 The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Molecular Cell 26: 41-50.


Schafer, T., B. Maco, E. Petfalski, D. Tollervey, B. Bottcher, U. Aebi, and E. Hurt. (2006). Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 441: 651-655.



Schafer, T., D. Strauss, E. Petfalski, D. Tollervey, and E. Hurt, 2003 The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22: 1370-1380.


Seiser, R. M., A. E. Sundberg, B. J. Wollam, P. Zobel-Thropp, K. Baldwin, M. D. Spector, and D. E. Lycan, 2006 Ltv1 is required for efficient nuclear export of the ribosomal small subunit in S. cerevisiae. Genetics 174(2): 679-691.


Siomi H, Matunis MJ, Michael WM, Dreyfuss G. 1993a. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res 21:1193–1198.


Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1994) Essential role for the KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell, 77, 33-39.


Spahn, C. M., R. Beckmann, N. Eswar, P. A. Penczek, A. Sali, G. Blobel, and J. Frank. (2001). Structure of the 80S ribosome from Saccharomyces Cerevisiae tRNA-ribosome and subunit-subunit interactions. Cell 107: 373-386.


Spahn, C. M., M. G. Gomez-Lorenzo, R. A. Grassucci, R. Jorgensen, G. R. Andersen, R. Beckmann, P. A. Penczek, J. P. Ballesta, and J. Frank, 2004 Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO Journal 23: 1008-1019.


Tindall K. R., and T. A. Kunkel. (1988). Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27: 6008-6013.

Warner, J. R., 1999 The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24: 437-440.