SNAPGENE FIND ORF DRIVER

Since it is not straightforward to monitor the rates of individual steps of translation 33, indirect measurements of protein expression are often necessary to infer the underlying mechanism of uORF-mediated regulation. For example, are some uORF regulatory mechanisms more effective than others at repressing protein expression across a wide range of biochemical parameters? How do uORFs alter the response of main ORF translation to changes in cellular and environmental conditions? Answering these questions requires a joint accounting of how the different steps of translation, such as initiation, scanning, and elongation, together influence the overall rates of uORF and main ORF translation. Further, interactions between scanning and elongating ribosomes on uORFs may cause dissociation of scanning ribosomes or enhanced initiation at start codons 23, 31, 32.ĭespite the plethora of proposed uORF regulatory mechanisms, their implications for the regulation of protein expression are not clear. Inefficient elongation can be driven by the nascent uORF peptide 27, 28, poorly translated codons in the uORF 29, 30, or small molecules such as amino acids or polyamines 23, 24. Although initiation rate usually limits translation, 18– 20 inefficient elongation or termination on uORFs can regulate translation by barricading scanning ribosomes from reaching the main ORF 21– 26. cerevisiae homolog of human ATF4) mRNA render main ORF translation sensitive to cellular levels of the eIF2α-GTP-tRNA Met ternary complex 16, 17. Multiple uORFs can interact together to regulate the re-initiation frequency at the main ORF. uORFs can constitutively repress translation by siphoning away scanning ribosomes from initiating at downstream main ORFs. UORFs can regulate translation via a variety of molecular mechanisms.

SNAPGENE FIND ORF DRIVER

For example, uORF mutations in oncogenes and tumor suppressors can act as driver mutations in cancer 14, 15.

uORF mutations are implicated in several human diseases via changes to main ORF translation 12, 13. Despite having poor initiation sequence contexts, many eukaryotic uORFs repress main ORF translation 1, 3, 4, 7- 11. uORFs can regulate gene expression via the biological activity of the uORF peptide, but they also often cis-regulate translation of the downstream main ORF 6, 7. Ribosome profiling studies estimate that at least twenty percent of these uORFs are actively translated 4, 5. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.Ībout half of human mRNAs have at least one upstream open reading frame (uORF) in their 5’ untranslated region 1– 3. We identify several human uORFs that repress main ORF translation via a similar terminal diproline motif. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF translation when ribosome loading onto the mRNA is reduced.

Here, we performed systematic measurements on the uORF-containing 5’ UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells.

However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. uORFs can potently regulate the translation of downstream open reading frames by several mechanisms: siphoning away scanning ribo-somes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. Upstream open reading frames (uORFs) are present in over half of all human mRNAs.

Snapgene find orf