Piero Carninci’s group at RIKEN published an article last year demonstrating that small RNAs can possess a large variety of 5′ cap structures including a 2,7 dimethylguanosine modification and found multiple cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in small RNA pools.


Source: DOI: 10.1371/journal.pone.0102895

This adds a further level of complication to small RNA sequencing library prep, as such protocols generally allow for sequencing only of RNAs with a 5′ monophosphate. In general, mature, functional microRNAs are thought to carry a simple 5′ monophosphate, and ligation kits such as the CleanTag™ Ligation Kit for Small RNA Library Preparation that use T4 RNA Ligase to fix oligonucleotides to the 5′ end of RNA will effectively oligocap this 5′-monophosphated RNA population. Carninci’s group used a variety of approaches including decapping with tobacco acid pyrophosphatase, CAGE, and immunoprecipitation with 2,2,7-trimethylguanosine to perform their study, but this isn’t the first time we have heard about cap structures on small RNAs.

Source: "MiRNA-biogenesis" by Narayanese at English Wikipedia. Licensed under CC BY-SA 3.0 via Commons - https://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpg#/media/File:MiRNA-biogenesis.jpg

Image Source: “MiRNA-biogenesis” by Narayanese, Wikipedia. 


We have known for quite some time that small RNAs can possess 5′ structures. The now discontinued ScriptMiner™ small-RNA–seq library prep kit from Epicentre® (an Illumina Company), for example, included protocols for removal of 5′ cap structures with Epicentre’s tobacco acid pyrophosphatase to allow capture of small 5′-capped and 5′-triphosphorylated RNAs in addition to 5′-monophosphate RNAs. Due to the discontinuation of tobacco acid pyrophosphatase, tebu-bio’s Decapping Pyrophosphohydrolase or CellScript’s Cap-Clip™ Acid Pyrophosphatase is now recommended to convert RNAs with diverse 5′ structures to 5′-monophosphate RNA for oligo-capping.

While next generation sequencing is quickly replacing array-based genomics/transcriptomics technologies, library prep for miRNA sequencing remains relatively complex. Due to the complexity of adapter-dimers and 5′ cap structures, array-based genome-wide miRNA expression analysis remains the first choice for many researchers.

The cap structures present on small RNAs appear to be either added during transcription or may be the result of a yet-to-be characterized mechanism for capping of RNA degradation products. An excellent resource for learning more about RNA 5′ structures is the Modomics website, which allows users to visualize various cap structures and gives the identify of the enzymes responsible for the modifications.

Source: http://modomics.genesilico.pl/pathways/pppN/

Source: http://modomics.genesilico.pl/pathways/pppN/


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