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Rockefeller University Researchers Find Skewed Expression of mRNA Components

NEW YORK (GenomeWeb) – Researchers from Rockefeller University this week reported that lopsided ratios in the expression of two parts of mRNA seems to correlate with fine tuning protein production.

After transcription, each mRNA molecule possesses a coding sequence that holds the instructions for protein production. The coding sequence is flanked by two sections that do not go on to make protein, the 3' untranslated region (UTR) and the 5' UTR. It has long been assumed that all three stick together within a single mRNA molecule.

But the researchers, whose findings were published in Neuron this week, have found that the popular theory is wrong. They found that UTR-to-coding-sequence-expression ratios in embryonic dopamine neurons was consistently high for many genes, including two in the Sox family. If this had been true in some embryos and not others, it wouldn't have been observable using these methods, Rockefeller University professor and principal investigator Mary Hynes told GenomeWeb.

The researchers used a technique called translating ribosome affinity purification (TRAP) that was developed at Rockefeller in 2008. It reveals translational profiles by isolating the genetic messages that govern protein production in different cell types. The mice the researchers used were engineered to express a specially tagged ribosome in only one particular cell type, so the researchers were able to look at genetic messages as they passed through the ribosomes.

For this study, the researchers used ventral tissue from dopamine-transporter and serotonin-transporter mice. They extracted mRNA from the tissue samples, purified it through the polysome purification process developed at Rockefeller, and then sequenced it. RNA was amplified with the SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit.

After sequencing, the gene transcripts were visually inspected using the Integrative Genomics Viewer (IGV) browser, which showed expression of 3' and 5' UTR and coding sequence regions. The researchers found that a number of genes in dopamine neurons showed abundant expression of 3' UTR mRNA sequences and little to no expression of the coding region of these mRNAs. This was true of two genes in the Sox family, Sox11 and Sox12, which are known to help differentiate cells during development.

This contradicted the common thinking that the 5' and 3' UTRs and the coding regions act as a united whole once an mRNA is transcribed. The researchers further decided to test the prevalence of the unbalanced ratio of UTR to coding sequence expression by examining 19 genes in situ. All the genes they examined, even those across different neurons/cells, showed similar expression patterns.

They also found that when they took protein expression into account for Map2 and tyrosine hydroxylase, the higher the ratio of expression of 3' UTR to coding sequence was, the lower the level of protein. "The [high UTR expression's] correlation with the protein regions was intriguing," said Hynes, adding that one can speculate that high levels of 3' UTR might somehow be involved in turning down the dial on protein production, though there is no evidence for that at the moment.  

Hynes and her colleagues plan to look at some of the biological reasons why this happens. Although it would be interesting to also explore the mechanism behind why this happens, she added, it's probably a project better suited for a bigger RNA lab.