Science meetings & conferences can be like geek Disneyland – speaking of which, let’s talk about POLYSOME PROFILING (a way to look at protein production) in terms of Disneyland trying to see how popular its different rides are. Instead of looking at how many people are on each ride, it looks at how many protein-making complexes called ribosomes are on each protein “recipe” copy (messenger RNA, mRNA).
This is NOT the same as ribosome profiling/footprinting, that technique we looked at yesterday. Polysome profiling tells you how popular a ride is (how many ribosomes are on an mRNA) at different times/under different conditions and/or how popular it is compared to other rides. Ribosome footprinting, on the other hand, tells you *where* people are along the ride’s course (where on the mRNA the ribosome’s bound) & whether & where they’re getting stuck (eek!). Let’s take a closer look.
The instructions for making proteins are in the form of messenger RNA (mRNA), and RNA/protein complexes called RIBOSOMES travel along them, inserting the corresponding amino acids (protein letters), which are brought to them by molecules called transfer RNAs (tRNAs). On one end of the tRNA is a 3-RNA-letter sequence called an anticodon that complements the 3-RNA-letter codon that the ribosome is currently standing on in the mRNA. On the other end of the tRNA is the corresponding protein letter (amino acid). When the tRNA brings the right amino acid, the ribosome helps transfer it to the growing chain and then moves on to the next codon, until it reaches a stop codon. Then, instead of tRNA, release factors bind and the amino acid chain (protein) gets released. This process called TRANSLATION and you can learn lots more about it here: http://bit.ly/2XwGdKO
But for now, just picture a ribosome moving along an mRNA and making a protein as it goes. Now, let’s make things more interesting… Let’s imagine that ribosome’s a boat and it’s traveling along the river in 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. A single boat on the ride is a monosome, and multiple boats on the same river is a polysome. When the boat reaches the end of the ride, instead of the reward of freedom, you get the reward of a freshly-made protein. In this scenario, we’ll imagine all the boats as being “full” (the number of people on them is the same) so we can use how many boats are moving through the ride as an indication of how popular the ride is.
The more popular the ride, the more boats on it -> More “popular” mRNAs (ones for proteins your cell really wants) will have more ribosomes on them, and make more protein. This is called TRANSLATIONAL REGULATION. But there’s a limit to how many you can fit.
So, if Disneyland decided it wasn’t such a small world after all – they couldn’t keep up with demands – they can open up more copies of the ride (make more mRNA). But building the ride (transcription) takes some time so it’s good to think ahead when possible for such TRANSCRIPTIONAL REGULATION. But not too far ahead because you don’t want a ride no one wants hogging resources you need to make other rides. Speaking of which…
It’s A Small World isn’t the only ride at Disneyland. So you can think of each “type” of mRNA (that is, mRNA for protein 1 or mRNA for protein 2) as different rides. So, let’s represent another mRNA (so instructions for making a different protein) as the Pirates of the Caribbean ride.
Similar in terms of setup – boats traveling through animatronic scenes – but they cater to different audiences. Older kids love it while the younger ones can find it frightening. So, demand for the different rides will change depending on who’s coming to Disneyland. So the different rides will have different popularities at different times – during the week during the school year, It’s a Small World was more popular, as kids too young for school (or missing just a couple days at least wasn’t as bad a thing) dominated, while, during Spring Break, when the older kids invaded, Pirates of the Caribbean was in higher demand.
Similarly, different proteins are needed at different times in a cell cycle. And in response to “special events” – like a new Pirates of the Caribbean movie coming out increasing demand for the Pirates ride, “special events” like responding to a hormone signal or getting a large shipment of sugar to break down can cause cells to change what proteins they make.
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
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. (Yep, these are sinky boats….)
Proteins and RNA absorb UV light, so a UV detector can scan the gradient and absorption peaks tell you where “stuff” is – and 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 and 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 and 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). This way, you don’t get fooled into thinking there’s more of an mRNA in the monosomal fraction just cuz you recovered that fraction’s RNA better.
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 the 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 and see how many copies get made.
A northern blot or qPCR work well if you know what recipe you’re looking for, but they’re low-throughput and you have to know what to look for. With the rise of high-throughput RNA sequencing methods, it’s now become possible to sequence the RNA associated with the different fractions (e.g. monosomal fraction, polysomal fraction).
Most of the time, when people talk about mRNA-seq, they’re typically talking about sequencing “all” of the mRNAs without addressing whether the RNAs are actually being actively translated. The idea is that if you break open a cell and count the number of mRNA copies of a gene there are, if you see a lot of an mRNA, a lot of its protein is likely getting made – but that’s an assumption that’s not always true. It’s easier since you don’t have to go through all of this ribosome fractionation, and there’s less risk of losing some of the RNA in the process, but you don’t know if that mRNA is actually being used.
This is different from ribosome footprinting, which we looked at yesterday. 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 protecting from cutting, so then you can release this protected RNA and sequence it to see where the ribosomes were. Since ribosome footprinting chews around the ribosomes, it separates ribosomes that are on the same mRNA (but different locations on it) at the same time. So what you end up seeing 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.
But, in the case of polysome profiling, since you don’t mess with the mRNA neighboring ribosomes are on, you do separate “3 on 1” from “2 and 1” and “1 and 1 and 1.” (But once you pass 8 or so on one you can’t tell if there are more cuz they all come out in the same fraction). But you can’t see where on the strand they are – so you lose information about whether certain regions are translated more slowly than others (rare codons causing a holdup?) or whether alternative start sites are being used.
So each technique has some pros and cons and they’re often both used, and hopefully you’re now a little less confused!
more on ribosome footprinting: https://bit.ly/ribosomefootprinting
more on some topics mentioned (and others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0