Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre‐mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell‐type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density‐gradient centrifugation, followed by RNA‐seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.

The generation of mature mRNA in the protozoan parasite Trypanosoma brucei requires coupled polyadenylation and trans splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3′ end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in T. brucei, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3′ end processing of mRNAs. Based on tandem-affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both in vivo polyadenylation and trans splicing, arguing for a general coupling of these two mRNA-processing reactions.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.