New details on how cytoplasmic RNA structures form

There are plenty of large mRNA agregates in cells. In the past few years, two of these structures have gained quite a bit of attention, Stress Granules (SGs) and Processing Bodies (PBs). mRNAs in SGs are loaded with 48S complex, which consists of the small ribosomal subunit, the cap binding complex (aka eiF4F) and the eIF3 complex. SGs are transient structures that are formed in cells experiencing stress such as arsenite, elevated temperatures and amino acid starvation. The key step in forming these structures is the inactivation of eIF2alpha, the protein that carries the initiator tRNA-methionine to the 48S complex. mRNAs that get shuttled into SGs are thought to be stored for future use.

PBs on the other hand are present in most cells constitutively (that means all the time). What's in PBs? All sorts of RNA degredation enzymes, including the miRNA machinery (aka the RISC complex), the nonsense-mediated decay proteins, decaping enzymes and the cytoplasmic exosome (the exosome that is responsible for RNA decay and not the unrelated exosome that is responsible for membrane trafficking). Bottom line: PBs are thought to be the place where mRNAs are destroyed.

Anderson's group, located right in our neighborhood, has seen that SGs and PBs seem to interact and possibly exchange material. From these observations they believe that mRNAs originally in SGs can be transported to interacting PBs. Such mRNAs would thus be targeted for destruction. Well to figure out how SGs and PBs are formed, the Anderson lab embarked on one of those siRNA screens and looked for proteins that were required for SG and/or PB assembly. So they treated cells with siRNAs then a couple of days later treated cells witrh arsenite to trigger SG formation and then visualize and score cells for the presence of these structures.

What did they find?

- Certain proteins were needed to form SGs and others for PBs indicating that you could have one type of structure without having the other. Interestingly this finding differs from some speculation by Roy Parker's group that examined the formation of PBs and a structure that resembles SGs in yeast. In that paper they propose that all mRNAs which end up in SGs must first go through PBs. The fact that Andersen's group can obtain cells that lack PBs but still form SGs in response to arsenite, suggests that mRNAs can enter into SGs without first visiting a PB. This finding also jibes with some of my results. In my paper from last year, I observed that certain transcripts lacking particular RNA elements, such as the SSCR, tend to accumulate into SGs but not in PBs. Once in the SGs, these mRNAs do not seem to be degraded but rather stored.

- Most members of the eIF3 complex and other translation initiation factors (eIF2B, eIF5) are required to form SGs. These results are consistent with the idea that mRNAs that are stalled at the 48S stage not only enter into these structures but catalyze their formation. Other hits included 9 subunits of the ribosome - I'm not sure if there are other homologs of these protens, but I would guess so seing that the ribosome is essential for viability. There are some other interesting hits in the paper that will be fodder for future studies.

- From some torturous reasoning, the Anderson group thought that cytosolic O-linked glycosylation may be important for SG formation. To their credit they proceeded to clearly demonstrate that the depletion of enzymes involved in producing GluNAc substrate (enzyme of the hexoseamine biosynthetic pathway), inhibited SG formation. Using an antibody against O-GluNAc glycosylated proteins the Anderson group localized some of the modified proteins to SGs, although most of the modified proteins seems to be nuclear. The nuclear stain did not change under stress conditions and I'm guessing that these modified proteins are likely to be transcription factors. Overall there was only a slight increase in O-GluNAc in cells treated with arsenite, however the majority of the proteins that are modified during stress were ribosomal in origin.

So what does this modification do?

Well most cell biologists have heard that proteins of the nuclear pore, especially FG-repeat containing proteins, get modified with O-GluNAc. Some have pointed out that OGluNAc and phosphorylation tend to be inversely correlated, leading some to conclude that one modification inhibits the other, although this theory is not widely held. Recently there has been quite a bit published on this post-translational modification. Here's a distilation of the current observations and theories:

1) The amount of O-GluNAc modified proteins increases when cells are deprived of amino acids or experience heat shock, two conditions that stimulate SG formation. I should also point out that there seems to be some connection between heat shock proteins, such as hsp70, and SG formation. More needs to be done to figure out exactly what's going on here.

2) O-GluNAc modification seems to protect cells from stress conditions. Tus when you knock down the hexsoamine biosynthetic pathway, worms die after exposed to stress.

3) The modification affects longevity in C elegans and seems to modify insulin signaling, a cell communication pathways with deep ties to stress response and longevity.

I have to say that it is still not quite clear why O-GluNAc modification is critical for initiating SG formation. Besides ribosomal proteins, some other proteins were found to be modified in response to arsenite such as RACK1 (a protein involved in programmed cell death) and prohibitin (a membrane bound protein that is thought to act as a chaperone in the mitochondrial matrix, but may lso play some unknown role out in the cytosol). Interestingly when prohibitin was depleated by siRNA treatment, SG formation was also inhibited.

Another puzzle is that proteins from both the small and large subunit were found to be modified in an arsenite dependent manner despite the fact that only the former are known to end up in SGs. This jibes with the idea that protein synthesis is deeply connected to SG formation, but the exact mechanistic logic is not quite clear to me.

Overall the paper is an interesting glimpse into the weird domain of RNA structures.

Ohn T, Kedersha N, Hickman T, Tisdale S, Anderson P.
A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly.
Nat Cell Biol. (08) 10:1224-31

For more on RNA granule-type structures check out these past post:
RNA Decay Particles
Review on RNA Granules
mRNA in dendrites: this message will self-destruct in 10 seconds
Still Confused about siRNA vs. miRNA?

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