Workshop Report 32

What do ncRNAs do? Mechanisms of action, functional relevance, importance of negative results

Gunter Meister, Biochemistry Center Regensburg, Regensburg University, Germany

Small regulatory ncRNAs are found in all higher eukaryotes. They play key regulatory roles in cellular processes that are as diverse as embryonic development, stress response or transposon silencing. All small ncRNA species are generated from longer precursor molecules and are finally incorporated into effector protein complexes. Of the small ncRNAs, recent research has focused on miRNAs that guide post-translational, temporary gene silencing. This is mediated by members of the so-called argonaute (AGO) protein family that bind to the miRNAs (Huntzinger and Izaurralde, 2011; Meister, 2013; Schraivogel and Meister, 2014; Dueck and Meister, 2014; Jonas and Izaurralde, 2015). Second, lncRNAs are being extensively studied. In mammals, lncRNAs are found in the nucleus and in the cytoplasm. In the nucleus, they mainly associate with chromatin and regulate the expression of specific genes. In the cytoplasm, they may bind to mRNAs thereby influencing post-transcriptional gene expression. LncRNAs can function as guides (enhancers of proper localisation of protein complexes), sponges (by binding to complementary RNAs thereby inhibiting their functionality), scaffolds (adaptors to bring two or more proteins into discrete complexes) or even nucleators for higher order chromatin structures (Ebert and Sharp, 2010; Salmena et al, 2011; Rinn and Chang, 2012). Similar to small RNAs, lncRNA expression is frequently altered in human diseases. Very recently, circular RNAs have been characterised (Hentze and Preiss, 2013). Under specific conditions or in specific tissues (e.g. the brain), circular RNAs can be abundant. They are generated by alternative splicing and may fulfil functions such as sponges for RNAs and proteins. Apparently, some circular RNAs even have coding potential and may contribute to the diversification of gene products. Finally, epitranscriptome, i.e. sugar or base modifications that are introduced after synthesis of the RNA transcript constitute procedures that, similar to epigenetics, exist ‘on top’ of coding and non-coding transcripts. Generally, RNAs may be modified at particular bases, as has been known for many years for ribosomal and transfer RNAs. Recently, it was found that such modifications are also present on mRNAs where they can influence gene expression. RNA-binding proteins may associate with transcripts, thereby changing their function independently of their sequence, and apparently RNAs can cross-talk by hybridisation (Lin and Gregory, 2014). All of these factors should be taken into account when analysing gene expression. Most likely, to date, only the ‘tip of the iceberg’ has been uncovered and many more such modifications with important biological functions remain to be detected.

Q&A

What triggers changes in gene expression? – These are complex mechanisms. At the molecular level, triggers could be transcription factors or modifying enzymes.

In terms of AOPs, what are the key issues one should focus on? – One should focus on the level of the transcript and determine if changes are sensitive. Very often, changes are small (e.g. five-fold increases of singular molecules), in which case they will be difficult to detect.