Scientists may have uncovered a new way for cells to regulate protein expression. In the December 16 Neuron, researchers led by Mary Hynes at Rockefeller University, New York, reported that in mouse neurons, the 3' untranslated regions of mRNAs were often expressed at higher or lower levels than the corresponding coding regions of the same strand. These expression patterns stayed consistent within particular neuronal subtypes and between animals, but varied with age and brain region, suggesting they were not random and might serve a purpose. Hinting at this, preliminary data on two genes linked high expression of the 3'UTRs to low expression of the encoded proteins. If the results hold up for more genes, this would add yet another wrinkle for geneticists trying to analyze gene expression.
Other researchers called the findings intriguing but preliminary. “This is a very interesting study which may give rise to new ways of thinking about the relationship of mRNA sequence to protein expression,” Bruce Yankner at Harvard Medical School wrote to Alzforum. Likewise, Philip De Jager at Brigham and Women’s Hospital, Boston, noted, “Often, the correlation between mRNA and protein levels is not very strong. This may explain part of the discrepancy we see.” However, both commenters noted that the sample size was limited, and suggested that researchers will need to look at many more genes in a systematic manner to determine if this represents a widespread regulatory phenomenon.
Hynes and colleagues first noticed the phenomenon serendipitously, while using translating ribosome affinity purification (TRAP) mice to study gene expression in developing dopamine neurons. These mice, developed by Nathaniel Heintz and colleagues at Rockefeller, allow researchers to selectively isolate mRNA from specific classes of neurons that express a particular promoter (see Heiman et al., 2008; Heiman et al., 2014). They do this by using a ribosome tag, such as enhanced green fluorescent protein/ribosomal subunit chimera, driven by that promoter. Researchers have used TRAP to examine RNA expression patterns from distinct subtypes of neurons, without having to isolate them first. This technology made the unequal mRNA expression patterns visible, Hynes told Alzforum.
Joint first authors Arif Kocabas and Terence Duarte used dopamine transporter promoter-based TRAP mice to isolate mRNA from mouse embryonic dopamine neurons. When they sequenced the mRNA, they noticed that for many transcripts there were thousands of copies of the 3'UTR, but virtually none of the coding region. To see if this was a common occurrence, the authors performed in situ hybridizations using probes to the coding and 3'UTR sequences of 19 genes, examining tissue from multiple brain regions and peripheral organs of mice, and from animals of different ages. The genes ranged from highly expressed housekeeping genes to transiently expressed developmental genes. In every case, the authors found unequal expression of coding regions and 3'UTRs at some ages and in some tissues. For each gene, the pattern stayed consistent across animals. In many cases, the authors saw precise spatial regulation, with adjacent subclasses of neurons possessing distinct expression patterns (see image above).
What purpose might these patterns serve? The authors compared mRNA to protein levels for two genes, tyrosine hydroxylase and microtubule-associated protein 2, for which they had good antibodies. For both genes, the authors found that the expression level of the protein product in specific tissues was not correlated with total mRNA levels, but instead with the ratio of coding sequence to 3'UTR. Cells with high levels of 3'UTRs expressed little to none of the corresponding protein product. The results hint that an excess of untranslated mRNA could help turn down unneeded protein expression, the authors suggest. Supporting this, an analysis of RNA sequencing data from the embryonic dopaminergic neurons revealed that the set of genes with high 3'UTR to coding region ratios was enriched for developmental genes, whose precise regulation is critical to development of various cells and organs.
Exactly how a cell acquires an excess of 3'UTR without its corresponding coding region remains unclear. A previous study by researchers led by John Mattick at the University of Queensland, Brisbane, Australia, found that 3'UTRs can be expressed separately from coding regions through post-transcriptional cleavage and degradation of the coding portions of the mRNA (see Mercer et al., 2011). These UTRs might function as noncoding RNA and regulate gene expression, the Australian group speculated. Hynes plans to further investigate this potential function by overexpressing 3'UTRs in cultured cells, and measuring whether this modulates protein expression. She will also examine more genes to determine if overexpression of UTRs consistently correlates with silencing.—Madolyn Bowman Rogers
- Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, Suárez-Fariñas M, Schwarz C, Stephan DA, Surmeier DJ, Greengard P, Heintz N. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 2008 Nov 14;135(4):738-48. PubMed.
- Heiman M, Kulicke R, Fenster RJ, Greengard P, Heintz N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat Protoc. 2014;9(6):1282-91. Epub 2014 May 8 PubMed.
- Mercer TR, Wilhelm D, Dinger ME, Soldà G, Korbie DJ, Glazov EA, Truong V, Schwenke M, Simons C, Matthaei KI, Saint R, Koopman P, Mattick JS. Expression of distinct RNAs from 3' untranslated regions. Nucleic Acids Res. 2011 Mar;39(6):2393-403. Epub 2010 Nov 12 PubMed.
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- Kocabas A, Duarte T, Kumar S, Hynes MA. Widespread Differential Expression of Coding Region and 3' UTR Sequences in Neurons and Other Tissues. Neuron. 2015 Dec 16;88(6):1149-56. PubMed.