Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.     

Small RNAs from plants,bacteria and fungi within the order Hypocreales are ubiquitous in human plasma

Background

The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Results

Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin. In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

Conclusions

A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-933) contains supplementary material, which is available to authorized users.