The Effect of Alternative Splicing Sites on Mirtron Formation and Arm Selection of Precursor microRNAs.

Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.

Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

The No-go decay system degrades plant mRNAs that contain a long A-stretch in the coding region.

RNA quality control systems identify and degrade aberrant mRNAs, thereby preventing the accumulation of faulty proteins. Non-stop decay (NSD) and No-go decay (NGD) are closely related RNA quality control systems that act during translation. NSD degrades mRNAs lacking a stop codon, while NGD recognizes and decays mRNAs that contain translation elongation inhibitory structures. NGD has been intensively studied in yeast and animals but it has not been described in plants yet. In yeast, NGD is induced if the elongating ribosome is stalled by a strong inhibitory structure. Then, the mRNA is cleaved by an unknown nuclease and the cleavage fragments are degraded. Here we show that NGD also operates in plant. We tested several potential NGD cis-elements and found that in plants, unlike in yeast, only long A-stretches induce NGD. These long A-stretches trigger endonucleolytic cleavage, and then the 5' fragments are degraded in a Pelota-, HBS1- and SKI2- dependent manner, while XRN4 eliminates the 3' fragment. We also show that plant NGD operates gradually, the longer the A-stretch, the more efficient the cleavage. Our data suggest that mechanistically NGD is conserved in eukaryotes, although the NGD inducing cis-elements could be different. Moreover, we found that Arabidopsis AtPelota1 functions in both NGD and NSD, while AtPelota2 represses these quality control systems. The function of plant NGD will be discussed.