RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells.

Background: Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amounts and are unlikely to be of functional importance. All RNA interference (RNAi) technology reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics. Results: In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resolution northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technology reagents. Conclusions: The conventional northern blotting enhanced to high resolution may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quantitative data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technology reagents expressed in cells.

Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.

Tandem repeats of various trinucleotide motifs are frequent entities in transcripts, and RNA structures formed by these sequences depend on the motif type and number of reiterations. The functions performed by normal triplet repeats in transcripts are poorly understood, but abnormally expanded repeats of certain types trigger pathogenesis in several human genetic disorders known as the triplet repeat expansion diseases (TREDs). The diseases caused by expanded non-coding CUG and CGG repeats in transcripts include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome. Another group of disorders in which transcripts containing translated CAG repeats play an auxiliary role in pathogenesis include Huntington’s disease and several spinocerebellar ataxias. In this review, we gathered existing knowledge regarding the structural features of triplet repeats in transcripts and discussed this in the context of various pathogenic mechanisms assigned to toxic RNA repeats. These mechanisms include aberrant alternative splicing, the inhibition of nuclear transport and export, induction of the innate immune response, alteration of a microRNA biogenesis pathway and abnormal activation of an RNA interference pathway. We also provide ideas for future investigations to reveal further mechanisms of pathogenesis directly triggered by mutant RNA repeats in TREDs.

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs.

MiRNAs are considered important players in oncogenesis, serving either as oncomiRs or suppressormiRs. Although the accumulation of somatic alterations is an intrinsic aspect of cancer development and many important cancer-driving mutations have been identified in protein-coding genes, the area of functional somatic mutations in miRNA genes is heavily understudied. Here, based on the analysis of large genomic datasets, mostly the whole-exome sequencing of over 10,000 cancer/normal sample pairs deposited within the TCGA repository, we undertook an analysis of somatic mutations in miRNA genes. We identified and characterized over 10,000 somatic mutations and showed that some of the miRNA genes are overmutated in Pan-Cancer and/or specific cancers. Nonrandom occurrence of the identified mutations was confirmed by a strong association of overmutated miRNA genes with KEGG pathways, most of which were related to specific cancer types or cancer-related processes. Additionally, we showed that mutations in some of the overmutated genes correlate with miRNA expression, cancer staging, and patient survival. Our study is the first comprehensive Pan-Cancer study of cancer somatic mutations in miRNA genes. It may help to understand the consequences of mutations in miRNA genes and the identification of miRNA functional mutations. The results may also be the first step (form the basis and provide the resources) in the development of computational and/or statistical approaches/tools dedicated to the identification of cancer-driver miRNA genes. This work was supported by research grants from the Polish National Science Centre 2016/22/A/NZ2/00184 and 2015/17/N/NZ3/03629.