Ribosome footprinting (aka profiling aka Ribo-seq) & polysome profiling

Ribosome footprinting (aka profiling aka Ribo-seq) & polysome profiling - an overview & comparison

Рет қаралды 3,378

2 жыл бұрын

When an mRNA becomes more popular, its RIBOSOME DENSITY (average # of ribosomes per mRNA for that gene) - (like average # of boats on each copy of the ride) increases. And so does the RIBOSOME OCCUPANCY - # of mRNAs of a gene bound by ribosomes (how many copies of the ride have boats on them). Note: Since different mRNAs are different lengths, and the longer the length, the more ribosomes can be bound at a time (but the longer it will take each to finish) you can take this into account - look at ribosomes per length unit when comparing
more on ribosome footprinting: bit.ly/ribosomefootprinting
more on polysome profiling: bit.ly/polysomeprofiling
How can we tell? POLYSOME PROFILING. How does it work? POLYSOME PROFILING looks at whether mRNAs are associating with full ribosomes and how many. The ribosome has lots of parts, but it has 2 main “pre-fab” “halves” - a small subunit & a large subunit. It needs both to be functional. Because the halves aren’t really halves, we can tell them apart by their weight. We can’t just stick them on a scale - they’re super tiny and surrounded with other molecules. Instead, we can use centrifugal separation to separate them in a sugar gradient.
After freezing the ribosomes in place - often with a chemical called cycloheximide, which halts elongation, you break open the cells (lyse them), remove the insoluble membrane parts, and add the cellular insides (cytoplasmic fraction) to a tube filled with a sugar gradient. And then you spin it really fast. The bigger half is denser so it will “sink” further. Both together will sink even further, only stopping when they reach that point in the sugar gradient where the sugar is as dense as it is.
And a cool thing is that the halves are “glued together” by mRNA binding - they don’t normally associate. So an “intact” ribosome implies there’s a recipe poised to be baked. A single (mono) full ribosome on an mRNA is called a MONOSOME. But usually, active bakeries have lots of bakers - POLYSOMES are multiple (poly) ribosomes attached to a single mRNA. And they weigh even more than the monosomes. So they’ll sink further.
Proteins and RNA absorb UV light, so a UV detector can scan the gradient and absorption peaks tell you where “stuff” is - & there are characteristic places in the gradient you can expect to find monosomes, polysomes, etc. You can detect “global” differences if the ratios are skewed (for example, if “all” translation is inhibited, you’d expect to see an increase in monosomes and a decrease in polysomes).
But the UV can’t tell you what specific RNAs are are in those peaks, so you can take the “fractions” of the gradient (e.g. the monsoonal fraction & polynomial fractions) & look to see what’s where. First you have to get the gradient out - after centrifugation & separation of your monosomes, polysomes, etc., you use some way to push the gradient out from a hole in the top of the tube (old school style by injecting a higher concentration of sucrose through the side of the tube to build pressure from the bottom or with a fractionation with a piston that pushes down from the top to squeeze it up through a hole in the piston. And you can UV it on the way out as you direct it into fractions (kinda like with protein chromatography except you’re taking the column with you & doing it from the bottom).
To do that you have to extract the RNA out of the sugar (often by phenol-chloroform extraction). It’s often harder to extract the RNA out of the goopier stuff (higher density sucrose), so you’re likely to lose more. To control for this, you often add known quantities of a control mRNA, like luciferase mRNA, to each fraction before you start trying to extract the RNA. This way, you can measure how much luciferase was lost during the extraction in each fraction to normalize the fractions (adjust them so they can be directly compared to one another).
But how do you know what’s in what fraction? If you have one mRNA in particular you’re interested in, you can see where that recipe ended up in a couple ways. One is by doing a northern blot on the various fractions. A northern blot is where you use electrophoresis to run RNA through a gel mesh which separates RNA pieces by size. And then you transfer those RNAs out of the gel and onto a membrane and use labeled probes complementary to RNA you’re looking for to see where that RNA is on the membrane. Alternatively, you can use RT-qPCR, which makes a bunch of copies of a region bookended by primers that you give it. So you can use primers specific to a gene of interest & see how many copies get made.
Ribosome footprinting lets you see where along the river the boats are at a certain point in time. Instead of leaving the mRNAs intact, you use RNases (RNA chewers) to cut up the RNA around the bound ribosomes - the region the ribosome is standing on (~30 letters) is protected so you can release this protected RNA & sequence it to see where ribosomes were.