What are the mechanisms and functions of cap-independent translation?
The primary pathway for eukaryotic translation initiation requires the concerted action of numerous protein factors that are specifically recruited to mRNAs possessing a 5’ m7G cap. In response to a variety of environmental perturbations, cells down-regulate cap-dependent translation while favoring expression of a select group of genes via alternative initiation mechanisms including direct recruitment of the translation machinery to internal ribosome entry sites (IRESs). We discovered a novel sequence-specific molecular mechanism that coordinately controls IRES-dependent translation of several yeast genes required for differentiation during starvation by using genetic and biochemical manipulations of both mRNA substrates and the translation machinery. We have now identified many new cellular IRESs (> 3 dozen and counting), and are currently working to determine their physiological roles and molecular architectures.
How does UTR diversity contribute to regulation of gene expression?
5’ untranslated regions (UTRs) influence translational efficiency of mRNAs through diverse mechanisms: long and/or structured 5’UTRs are more difficult to scan, yet may afford resistance to 5’ to 3’ exonucleases; specific sequence or structure elements provide internal recruitment sites for translation factors or regulatory proteins; and upstream open reading frames (uORFs) modulate translation initiation site choice and efficiency. Alternative 5’UTR production is ubiquitous in eukaryotes and has enormous regulatory potential, perhaps comparable to that afforded by alternative splicing (which may also serve to increase 5’UTR diversity). We are combining high-throughput 5’end sequencing with translation-state profiling to obtain comprehensive knowledge of 5’UTR utilization under various growth states. We will use this rich dataset to propose and then test the ‘rules’ that govern translational efficiency in vivo.
Are ribosomes specialized?
Ribosomes are magnificent and versatile molecular machines. Although gorgeous high-resolution structures tempt us to regard ribosomes as unchanging homogeneous entities, many opportunities for molecular specialization exist, including alternative ribosomal protein isoforms, ribosome accessory proteins, post-translational modification of ribosomal proteins, and post-transcriptional modification of ribosomal RNAs. We are taking a proteomics approach, in collaboration with Steve Carr’s group at the Broad Institute, to investigate the changes in ribosome composition that are associated with cellular adaptive responses. We are exploring the functional consequences of ribosome specialization through translation-state profiling and biochemical characterization of mutant (forcibly unspecialized) ribosomes.
Carlile, T. M., Rojas-Duran, M. F., Shin, H, Zinshteyn, B., Bartoli, K. B., and Gilbert, W. V. (2014). “Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells”. Nature, in press. Highlighted in Nature Reviews Genetics. Highlighted in Nature Methods.
Vaidyanathan, P. V., Zinshteyn, B., Thompson, M. K., and Gilbert, W. V. (2014) “Protein kinase A regulates gene-specific translational adaptation in differentiating yeast”. RNA 20 (6): 912-922.
Zinshteyn, B. and Gilbert, W.V. (2013) “Loss of a Conserved tRNA Anticodon Modification Perturbs Cellular Signaling”. PloS Genetics 9 (8): e1003675.
Arribere, J. A. and Gilbert, W. V. (2013) “Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing”. Genome Research 23 (6): 977-87.
Rojas-Duran, M. F. and Gilbert, W. V. (2012) “Alternative transcription start site selection leads to large differences in translation in yeast”. RNA 18 (12): 2299-305.
Arribere, J. A., Doudna, J. A., and Gilbert, W. V. (2011). “Reconsidering movement of eukaryotic mRNAs between polysomes and P-bodies”. Molecular Cell 44 (5): 745-58. Highlighted by Faculty of 1000.
Gilbert, W. V. (2011). “Functional specialization of ribosomes?” Trends in Biochemical Sciences 36 (3): 127-32.
Gilbert, W. V. (2010). “Alternative ways of thinking about cellular internal ribosome entry”. Journal of Biological Chemistry 285 (38): 29033-8.