Proud parents might show you pictures of baby footprints – proud translation researchers might show you pictures of RIBOSOME FOOTPRINTS. I’ve seen many such “mementos” at the virtual RNA meeting I’m at, so I thought a quick review was warranted. Ribosomes are protein-making complexes and instead of looking to see if they’ve grown, scientists look to see what they’re “standing on” to tell what proteins are being made and how efficiently, using a process called ribosome footprinting (aka ribosome profiling or Ribo-Seq).
“Translational research” typically refers to research that, unlike “basic research” which is “just” aimed at finding out how things work, has direct applications. The classic example is how basic science on a bacterial immune defense mechanism led to the translational application of CRISPR-Cas gene editing. But, at the RNA meeting, “translational research” has a more basic meaning as well – research on TRANSLATION!
TRANSLATION is the process of making a protein (polypeptide). An RNA/protein complex called the RIBOSOME travels along a protein’s instructions (which are written in the language of RNA, with its A, U, C, & G nucleotide letters) and “Translates” that messenger RNA (mRNA) into the protein language of amino acids.
The protein alphabet is bigger than the RNA/DNA one – instead of just 4 letters, the protein alphabet has 20 (common) letters, so you need 3 RNA letters (1 codon) to spell one protein letter. The ribosome travels along the mRNA in codon-long steps and transfer RNA (tRNA) brings it the corresponding amino acids to be added to the growing chain through peptide bonds. (the tRNA has an anticodon that matches the codon the ribosome’s standing on and is “charged” with the corresponding amino acid letter to pass off to the growing chain with the help of the ribosome).
The ribosome isn’t allowed access to the original copy of the instructions – those are too valuable because if something gets messed up those mutations can get passed on to future cells. And if the mutation happens in cells of the germ line, whose future cells can go on to make other people, that mutation can get passed on to those future people too. So the cells lock the originals up in a membrane-bound compartment of the cell called the NUCLEUS and only let out RNA copies, which are made in a process called transcription.
Those originals are written in DNA, not RNA. It’s a lot like RNA except it has one less oxygen in its backbone’s ribosome sugar (hence the D for Deoxyribose) and that makes it more stable. So it’s locked up AND in “pen” instead of “pencil.” But it has almost the same “unique letter” parts (nitrogenous bases) – both have A, C, & G, but DNA has T instead of U. These letters are just groups of atoms that your cells keep stocks of & combines in different orders, kinda like old-school typesetting.
You have lots of copies of each letter, but you don’t have the same number of each (in this case it doesn’t make sense to have the same # of q’s & a’s). So, sometimes, if the protein has a lot of a less-common letter, especially in a row, it can stall while it waits for the cell to “check the stockroom” – so certain regions of mRNA can be translated more slowly. If a ribosome gets super stuck, a process called no-go decay can be triggered & the cell’s ribosome quality control systems come to the rescue. Even when the stuckness isn’t quite so dramatic, regions can be translated more slowly for other reasons, and this can serve as an important regulatory mechanism.
How do scientists know this? They can use something called RIBOSOME FOOTPRINTING (aka ribosome profiling).
Time for another weird analogy. Imagine the “It’s a Small World” Disneyland ride. For those lucky ones who may not be familiar, it’s a ride where groups of people travel on boats on a river through a series of animatronic scenes depicting various places around the world with a super annoying song playing.
You can think of the river as the mRNA and the boats are the ribosomes. And ribosome footprinting lets you see where along the river the boats are at a certain point in time. In this nightmare you don’t just have one of the rides. You have lots of copies of them. So, what this experiment shows you is the average of where the boats are in all those copy “rivers.” So you can’t tell if you have 3 boats on the same river or 2 on 1, and 1 on another, 1 each on 3 etc. And there are probably a lot more of these, because actively translating mRNA are usually associated with POLYSOMES, meaning that there are lots of individual ribosomes (MONOSOMES) on them.
Since you’re seeing the average, if there’s a slow region of the river, you’ll see a higher average occupancy, whereas if it’s smooth sailing all the way you’d see a more even spread.
How it works is you take cells (often cells in different conditions you want to see if are affecting things) then you add cyclohexamide – this shuts down the ride and then you look to see what RNA each ribosome hides! Cyclohexamide is a translation inhibitor, so the ribosomes get stuck and you can see where they get stuck. (though cyclohexamide can cause some artifacts so newer methods are avoiding it and instead using different chemicals or ditching the chemicals and just flash-freezing them).
After stopping the ride, you want to separate all the ribosomes. And you want to just see exactly where they are, not just the “general vicinity” (you want GPS level, not “go till you see the tree and turn right then walk a few steps”) The ribosome protects the mRNA it’s bound to (~30nt), but not the surrounding mRNA. So you can introduce RNA “scissors” (nucleases) to cut up the mRNA. The mRNA not bound by ribosomes will get chewed up, but the mRNA the ribosome’s “sitting on” will stay safe. Then you can release these saved bound parts. And sequence them.
The cutting step is called an RNAse protection assay. RNAse Unlike the “restriction endonucleases” or “restriction enzymes” we often use to cut & past DNA together at precise sites, here we want promiscuous scissors so it will chew around all the ribosomes, not just ones that happen to be next to a “code word.” But not so promiscuous that they chew up the RNA part of the ribosome (proteins often get all the credit, but most of the grunt work of the ribosome actually comes from its ribosomal RNA (rRNA components).
Before you do the sequencing, you need to isolate the ribosomes so that you’re only sequence the RNA that was actually being stood on, not the cut off pieces, nor pieces of RNA doing other things. This isolation can be done in several different ways including ultracentrifugation-based techniques where you basically spin the mixture in a dense sugar gradient to separate things by size or using purification columns that are coated with antibodies that bind to ribosomes but not other things.
The sequencing part will tell you where the ribosome was standing – getting the sequences generally is done by first adding adapters to the little RNA pieces and reverse transcribing them into DNA, which is more stable and copy-able using PCR. These sequences are then fed into a computer program that aligns them to all the cell’s recipes to tell what specific protein recipes those sequences are part of. And not just which recipe, but WHERE on the recipe. If you see a bunch in the same part of a recipe, for example, it could indicate that translation is slow in that region (possibly due to something like a rare codon that there are fewer tRNAs for, so it takes longer for that tRNA to find its way there).
You can take “where” one step further with another “add-on” – initiation site profiling can be used to find the translation start sites (some mRNAs actually have several “alternative start sites”). This start-finding can be done using a drug called harringtonine, which only stops translation at the first step.
You can get even more information if, in addition to sequencing the footprints, you sequence ALL the mRNA – a process typically referred to as RNA-seq. This can tell you the relative abundance of each mRNA and you can then compare it to how many ribosomal footprints you got from that mRNA to get an idea of how “efficiently” that mRNA is being made. You need to know the total copies because if you find a bunch of bound ribosome-bound fragments from an mRNA that could represent a few, highly translated, mRNAs or lots of lowly translated mRNAs.
Going back to the It’s a Small World analogy, it’s kinda like the difference between having a ton of It’s a Small World rides but only a few boats are traveling each versus having a few of the rides but lots of boats on each. (This can also be distinguished using a technique called polysome profiling which looks at “boat abundance”)
more on some topics mentioned (and others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0