New & Noteworthy

Pseudouridine: Not Just for Noncoding RNA Anymore

September 11, 2014

If you think back really hard to your basic molecular biology classes you can probably remember that weird nucleotide pseudouridine (ψ). You probably learned that it is found in lots of tRNAs and rRNAs but never in mRNA. You also may remember that while its function is still a bit unclear, it may have something to do with RNA stability and/or helping aminoacyl transferases interact with tRNAs.

Like this monument to Stalin that was dynamited in 1962, old dogmas like pseudouridine’s absence from mRNA are being cleared away with help from yeast. Images from Wikimedia Commons

If a new paper in Nature holds up, one of those things we learned is almost certainly wrong. In this study, Carlile and coworkers show pretty convincingly that ψ is also found in mRNA. And not only that, but it may be doing something important there.

The authors used a sensitive high-throughput technique called Pseudo-seq to look for ψ in all the RNA in a yeast cell. The first step in this technique is to treat the RNA with a chemical called CMC.* This chemical reacts with ψ in such a way as to create a block to reverse transcriptase. In other words, reverse transcriptase can only convert RNA into DNA up to the point of a ψ. The next step is to analyze the products and to determine where reverse transcriptase has been halted. 

Carlile and coworkers first validated their technique by looking at RNAs known to have ψ’s. They showed that their technique had an estimated false discovery rate of 5% for highly expressed genes and 12.5% for poorly expressed genes. They were now ready to tackle mRNA to see what they could find.

They first looked at the mRNA of the yeast Saccharomyces cerevisiae during post-diauxic growth (after log phase) and found 260 ψ’s in 238 protein coding transcripts. This is 260 more ψ’s than had been found before.

The next step was to try to get a feel for whether or not these changes were important. To do this, they decided to compare pseudouridylation (we promise not to use that word again!) in log phase and post-diauxic growth. They found that 42% of the sites modified after log phase were not modified during log phase. In other words, it looks like the level of mRNA modification is different depending upon the growth rate. 

Uracils are modified to ψ by a surprisingly large number of enzymes. One enzyme, Cbf5p, uses snoRNA guide sequences to find the right uracils to modify. Cbf5p may not be that important for converting U’s to ψ’s in mRNA , however, since only 3/260 of the sites identified by the authors appeared to be targeted by this enzyme.

E. coli pseudouridine synthase. Image from Wikimedia Commons

The other nine known enzymes in yeast all have the rather unfortunate acronym “PUS,” for PseudoUridine Synthase. Carlile and coworkers tested the effects of individually deleting eight of these on their newly identified ψ sites in mRNA and found that deleting PUS1 affected the highest number of mRNAs.  Interestingly, many of the Pus1p target sites were modified more often during post-diauxic growth than during exponential growth. Deleting the other PUS genes had similar, if smaller, effects. 

The authors next confirmed that something similar happens in human cells. Using very strict criteria, they identified 96 ψ’s in 89 human mRNAs and found that some of these were regulated by growth conditions (serum starvation), just as in yeast. So, modification of mRNAs with this interesting residue appears to happen in people too (or at least in HeLa cells).

Finding ψ’s in mRNA is a big contradiction to everything we’ve been taught!  The next step is to figure out what they are doing there, and there are lots of possible answers.

One possibility is that the newly discovered mRNA modifications make possible a whole new set of translated proteins. Adding a ψ to mRNA changes codon usage at that position in vitro. For example, one study found that converting the stop codons UAA and UGA to ψAA and ψGA, respectively, changed them from stop codons into sense codons both in vitro and in vivo. So ψ’s in mRNAs could cause a whole slew of new alleles to appear under certain conditions – at the RNA level instead of the DNA level. A proteomics study should help determine whether this is happening or not.