A recent post described small Open Reading Frames (smORFs) and DWarf Open Reading Frames (DWORFS) — see the link at the bottom. Now it’s time for uORFs (upstream Open Reading Frames). Upstream of what you might ask? Well messenger RNA is grabbed by the ribosome at one end (called the 5′ end). The current thinking was that the ribosome marched along the mRNA from the 5′ to the 3′ direction looking for the sequence Adenine Uridine Guanine (AUG) which codes for methionine. It then begins reading the mRNA 3 nucleotides at a time and tacking amino acids onto the methionine. This is called translating mRNA into protein. What about the 5′ end of the mRNA before the AUG is reached (perhaps hundreds of nucleotides later) — it isn’t translated which is why its called the 5′ UTR (5′ UnTranslated Region). In bacteria its only a few nucleotides, but our 5′ UTRs can have thousands — https://en.wikipedia.org/wiki/Five_prime_untranslated_region.
Two other terms of art are upstream and downstream. Since the ribosome flows from 5′ to 3′ on mRNA, any nucleotide 5′ to a given point is called upstream, and anything 3′ is called downstream. Logical terminology — what a pleasure.
So a uORF is an upstream Open Reading Frame. Upstream to what? Why to the AUG (the initiator codon). The assumption had always been that since there was no initiator AUG codon on this region — that proteins couldn’t be made from the uORF. Wrong.
This is where [ Science vol. 351 p. 465 aad2867 – 1 –> 9 ’16 ] comes in. It turns out that the ribosome can translate some of these uORFs in protein, and the paper describes a clever technique (called 3T) they developed to find them. One of the problems in finding uORF proteins is that some are quite small, and are missed in the usual protein assays. One uORF from ATF4 contains only3 amino acids which is so small that mass spectrometry can’t see it.
The paper makes the amazing statement that — Nearly half of all mammalian mRNAs harbor uORFs in the 5′ UTRs, and many are initiated with nonAUG start codons. They may be a general mechanism to regulate downstream coding sequence expression and gives two citations that I must have missed in my reading .
For instance Binding immunoglobulin Protein (BiP aka Heat Shock Protein family A member 5 – HSPA5 ) contains uORFs exclusively initiated by UUG and CUG start codons (not AUG).
What might the functions of uORF actually be? The obvious one is that the proteins made from them might actually be doing something. What could a 3 amino acid protein possibly do? Lots. Consider thyrotropin releasing hormone which helps control your thyroid — it is pyroglutamic acid histidine proline. Then there is met-encephalin which has 5 amino acids and is one of the endogenous opiate peptides your brain uses.
Another possibility is that just translating the uORF into protein controls the translation of the protein starting with the AUG codon. This isn’t so far fetched. A recent paper [ Nature vol. 529 pp. 551 – 554 ’16 ] gave a 3 dimensional structure for RNA polymerase II transcribing a DNA template into mRNA. The authoress (Carrie Bernecky) was kind enough to supply the dimensions of the complex when I wrote her. Remember you can consider the DNA double helix as a cylinder 20 Angstroms in diameter. It is roughly 150 x 150 x 160 Angstroms. Figuring 3 stacked nucleotides/10 Angstroms, this is enough to obstruct 45 nucleotides of DNA upstream of the actual start site.
This is just another example of room at the bottom, where all sorts of small molecule metabolites, small RNAs, small DNAs are just being unearthed and their structure determined. For more on this please see the following link