Next-generation sequencing has revolutionized clinical diagnostic testing. Yet, for a substantial proportion of patients, sequence information restricted to exons and exon-intron boundaries fails to identify the genetic cause of the disease. Here we review evidence from mRNA analysis and entire genomic sequencing indicating that pathogenic mutations can occur deep within the introns of over 75 disease-associated genes. Deleterious DNA variants located more than 100 base pairs away from exon-intron junctions most commonly lead to pseudo-exon inclusion due to activation of non-canonical splice sites or changes in splicing regulatory elements. Additionally, deep intronic mutations can disrupt transcription regulatory motifs and non-coding RNA genes. This review aims to highlight the importance of studying variation in deep intronic sequence as a cause of monogenic disorders as well as hereditary cancer syndromes.

We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human β-globin genes that are either normal or defective in splicing or 3Ј end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, α-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human β-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3Ј end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3Ј end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.

In eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein in the cytoplasm requires precise and extensive modification of the nascent transcript. Any failure that compromises the integrity of an mRNA may cause its retention in the nucleus and trigger its degradation. Multiple studies indicate that mRNAs with processing defects accumulate in nuclear foci or ‘dots’ located near the site of transcription, but how exactly are defective RNAs recognized and tethered is still unknown. Here, we present evidence suggesting that unprocessed β-globin transcripts render RNA polymerase II (Pol II) incompetent for termination and that this quality control process requires the integrity of the nuclear exosome. Our results show that unprocessed pre-mRNAs remain tethered to the DNA template in association with Pol II, in an Rrp6-dependent manner. This reveals an unprecedented link between nuclear RNA surveillance, the exosome and Pol II transcriptional termination.

ABSTRACT

The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA‐1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA‐1 to consensus sites in the beta‐globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA‐1 foci seen by immunofluorescence. The GATA‐1 nuclear bodies differed from previously described nuclear structures and there was no co‐localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c‐Jun and TBP) or haemopoietic (NF‐E2) transcription factors. Interestingly, GATA‐2 and GATA‐3 proteins also localized to the same nuclear bodies in cell lines co‐expressing GATA‐1 and −2 or GATA‐1 and −3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein‐protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.

Eukaryotic cells have a surveillance mechanism that identifies aberrantly processed pre-mRNAs and prevents their flow to the cytoplasm by tethering them near the site of transcription. Here we provide evidence that mRNA release from the transcription site requires the heptad repeat structure of the C-terminal domain (CTD) of RNA polymerase II. The mammalian CTD, which is essential for normal co-transcriptional maturation of mRNA precursors, comprises 52 heptad repeats. We show that a truncated CTD containing 31 repeats (heptads 1–23, 36–38, and 48–52) is sufficient to support transcription, splicing, cleavage, and polyadenylation. Yet, the resulting mRNAs are mostly retained in the vicinity of the gene after transcriptional shutoff. The retained mRNAs maintain the ability to recruit components of the exon junction complex and the nuclear exosome subunit Rrp6p, suggesting that binding of these proteins is not sufficient for RNA release. We propose that the missing heptads in the truncated CTD mutant are required for binding of proteins implicated in a final co-transcriptional maturation of spliced and 3′ end cleaved and polyadenylated mRNAs into export-competent ribonucleoprotein particles.

Transcription and splicing are fundamental steps in gene expression. These processes have been studied intensively over the past four decades, and very recent findings are challenging some of the formerly established ideas. In particular, splicing was shown to occur much faster than previously thought, with the first spliced products observed as soon as splice junctions emerge from RNA polymerase II (Pol II). Splicing was also found coupled to a specific phosphorylation pattern of Pol II carboxyl-terminal domain (CTD), suggesting a new layer of complexity in the CTD code. Moreover, phosphorylation of the CTD may be scarcer than expected, and other post-translational modifications of the CTD are emerging with unanticipated roles in gene expression regulation.