Understanding how ribosomes choose translation initiation sites on mRNAs is an ongoing challenge in biology and is of great importance for investigation of cell function. We test the hypothesis that sequences just downstream of the translation start codon of mRNAs base pair with a conserved sequence in 18S rRNA termed the 530 loop that is located in the ribosome entrance tunnel for mRNAs. We propose that this base pairing helps position the AUG start codon at the ribosome P site facilitating translation initiation. This hypothesis is based on the observation that the 530 loop has base-pair complementarity to a three-nucleotide periodicity found in protein open reading frames and this periodicity is significantly enhanced 10-20 nucleotides downstream of the start codon of yeast proteins detected by peptide MS/MS as well as TAP-tagged proteins exhibiting high expression in large-scale western analysis. Using CRISPER-Cas9 gene editing, we will change the mRNA sequences 10-20 nucleotides downstream of the start codon to sequences that are not expected to base pair with the 530 loop and we will test using western analysis whether translation is depressed as predicted by the model. We will also test sequences that change the reading frame registration of the base pairing to assess whether the precise positioning of the base-paired mRNA is important for translation initiation. Enhancement of the same three-nucleotide periodicity is also found 10-20 nucleotides after non-standard start codons that initiate translation upstream or downstream of the annotated start. We will test whether reducing potential for base pairing by these sequences to the 530 loop down-regulates initiation at these non-standard start sites. Improving our understanding of translation initiation will be important in studying gene expression particularly in contexts where altered expression leads to gene disfunction, such as in disease states.

Public Health Relevance

We are investigating a core question in biology: how ribosomes choose translation start sites on mRNAs focusing in particular on the hypothesis that mRNA sequences just after the start codon base pair with a conserved sequence of rRNA in the ribosome's mRNA entrance tunnel thereby helping to position the ribosome for the start of translation. Improving our understanding of the control of protein translation and the resulting repertoire of proteins in the proteome, will be useful for interpretation cell functions and mRNA sequence variants and mutations associated with disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM120719-01
Application #
9171027
Study Section
Special Emphasis Panel (ZRG1-GGG-F (80)A)
Program Officer
Bender, Michael T
Project Start
2016-09-01
Project End
2019-08-31
Budget Start
2016-09-01
Budget End
2019-08-31
Support Year
1
Fiscal Year
2016
Total Cost
$491,599
Indirect Cost
$192,391
Name
Wesleyan University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
145683954
City
Middletown
State
CT
Country
United States
Zip Code
06459