Advances in sequencing technology have allowed scientists to peer inside living cells and follow the fate of small RNAs. The expedition has uncovered new mechanisms concerning small RNA production, their assembly into gene-regulatory complexes, and their decay, as reported in the current issue of Molecular Cell.

While historically the cellular role of RNA was associated with protein production, we now know of a plethora of small RNA fragments that are not translated into proteins but fulfil alternative functions in every cell of our body. Among these are microRNAs (miRNAs) that play key roles in many biological processes: they are universal regulators of gene expression in many different biological settings – from organism development to physiological responses – and interact with oncogenes and tumor suppressor genes, making them promising players in cancer research. Despite their tiny size, the impact of miRNAs on cell biology and disease mechanisms is huge, and their discovery more than 20 years ago launched a whole new field of research. 
Elucidating the molecular processes governing their production, processing and regulation is key to understanding miRNA function. Easier said than done: obtaining such information requires scientists to look inside living cells and observe the molecular fate of miRNAs from their birth to their death. Now a novel technology for time-resolved transcriptomics (SLAMseq) developed in the Ameres lab at IMBA opens the door – or the cell – to acquire unprecedented insights into this black box in small RNA biology.  

SLAMseq is an innovative sequencing method that allows nascent RNA to be distinguished from “old” RNA by metabolic labeling and chemical nucleoside conversion. Using SLAMseq, a team of scientists from the Vienna BioCenter recently succeeded in characterizing the transcriptional response of cancer genes in a highly time-resolved manner. Now they have applied their technology to investigate the ‘lifecycle’ of microRNAs from their birth by rapid production to changes in composition as they age. 

How small RNAs get to work

“We were surprised to see that most miRNAs were transcribed and processed much faster than what was previously observed for protein-coding transcripts. But not only that! They also rank among the longest-lived transcripts that are found in cells,” says Veronika Herzog, who is a postdoc at IMBA and the co-first author of the current publication. 
This combination of rapid production and unusual stability enables miRNAs to accumulate to extraordinary levels, reaching up to a hundred thousand of copies inside one cell! These findings reconcile previous studies which suggested that miRNAs do not bind very strongly to targets; the enormous abundance of miRNA ensures that their targets are constantly occupied to ensure the effective regulation of gene expression. But what limits miRNA function inside cells? In this paper scientists show that the sparse number of available Argonaute proteins (which bind to miRNAs to effect gene silencing) ensures high fidelity in gene regulation; this is because other RNA species are prevented from accidentally `hooking up’ with an empty argonaut protein to create an errant gene regulatory unit.

MicroRNAs shrink as they age

Like most transcripts, also miRNAs must be turned off after they fulfilled their function. “In fact, miRNAs exhibit very precise spatiotemporal expression patterns. That means that defined small RNA repertoires must be established and dismantled in a controlled manner, for example during cellular differentiation and organismal development,” explains Brian Reichholf, co-first author of the paper. But what makes miRNAs disappear? When they looked at the stability of individual miRNAs, the scientists noted a broad range of half-lives between classes. The team also found that the composition of miRNAs changes as they age: Nucleotides are trimmed from one end and removed. Interestingly, unstable miRNAs shrink more quickly than stable miRNAs, exposing a molecular signature of small RNA turnover. They could also attribute this shrinking to an enzyme called Nibbler, which may be a novel player in the regulation of miRNA turnover.

In contrast to miRNAs, other classes of small RNAs were not subjected to such a molecular aging. Among these were small interfering RNAs (siRNAs), that many organisms – such as fruit flies – use to defend themselves against virus infections. Compared to miRNAs, where expression patterns need to be controlled in a precise manner, siRNAs seem to be resistant to molecular shrinking. As a consequence, they are also stable than miRNAs, which may benefit a long-lasting and effective antiviral response. 

It seems that this fascinating story is just the beginning: “The ability to monitor the dynamics of gene expression is a fascinating emerging area of research. With our novel technology, we are able to bridge temporal resolution gaps, to get novel insights into the processes that bring our genome to life, “says Stefan Ameres, group leader at IMBA.

Original Publication: Reichholf, Herzog et al., “Time-resolved small RNA sequencing unravels the molecular principles of microRNA homeostasis”, Molecular Cell

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