The nucleoplasm is not homogenous; it consists of many types of nuclear bodies, also known as nuclear domains or nuclear subcompartments. These self-organizing structures gather machinery involved in various nuclear activities. Nuclear speckles (NSs) or splicing speckles, also called interchromatin granule clusters, were discovered as sites for splicing factor storage and modification. Further studies on transcription and mRNA maturation and export revealed a more general role for splicing speckles in RNA metabolism. Here, we discuss the functional implications of the localization of numerous proteins crucial for epigenetic regulation, chromatin organization, DNA repair and RNA modification to nuclear speckles. We highlight recent advances suggesting that NSs facilitate integrated regulation of gene expression. In addition, we consider the influence of abundant regulatory and signaling proteins, i.e. protein kinases and proteins involved in protein ubiquitination, phosphoinositide signaling and nucleoskeletal organization, on pre-mRNA synthesis and maturation. While many of these regulatory proteins act within NSs, direct evidence for mRNA metabolism events occurring in NSs is still lacking. NSs contribute to numerous human diseases, including cancers and viral infections. In addition, recent data have demonstrated close relationships between these structures and the development of neurological disorders.

Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

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.

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.

A growing body of evidence indicates that miRNAs may either drive or suppress oncogenesis. However, little is known about somatic mutations in miRNA genes. To determine the frequency and potential consequences of miRNA gene mutations, we analyzed whole exome sequencing datasets of 569 lung adenocarcinoma (LUAD) and 597 lung squamous cell carcinoma (LUSC) samples generated in The Cancer Genome Atlas (TCGA) project. Altogether, we identified 1091 somatic sequence variants affecting 522 different miRNA genes and showed that half of all cancers had at least one such somatic variant/mutation. These sequence variants occurred in most crucial parts of miRNA precursors, including mature miRNA and seed sequences. Due to our findings, we hypothesize that seed mutations may affect miRNA:target interactions, drastically changing the pool of predicted targets. Mutations may also affect miRNA biogenesis by changing the structure of miRNA precursors, DROSHA and DICER cleavage sites, and regulatory sequence/structure motifs. We identified 10 significantly overmutated hotspot miRNA genes, including the miR-379 gene in LUAD enriched in mutations in the mature miRNA and regulatory sequences. The occurrence of mutations in the hotspot miRNA genes was also shown experimentally. We present a comprehensive analysis of somatic variants in miRNA genes and show that some of these genes are mutational hotspots, suggesting their potential role in cancer.