The changing paradigm of intron retention: regulation, ramifications and recipes

Intron retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, insects and viruses. It was generally assumed that mis-splicing, leading to the retention of introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs

Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease

Holding on to junk bonds: intron retention in cancer and therapy

Intron retention (IR) in cancer was for a long time overlooked by the scientific community, as it was previously considered to be an artifact of a dysfunctional spliceosome. Technological advancements made in the last decade offer unique opportunities to explore the role of IR as a widespread phenomenon that contributes to the transcriptional diversity of many cancers. Numerous studies in cancer have shed light on dysregulation of cellular mechanisms that lead to aberrant and pathologic IR. IR is not merely a mechanism of gene regulation, but rather it can mediate cancer pathogenesis and therapeutic resistance in various human diseases. The burden of IR in cancer is governed by perturbations to mechanisms known to regulate this phenomenon and include epigenetic variation, mutations within the gene body, and splicing factor dysregulation. This review summarizes possible causes for aberrant IR and discusses the role of IR in therapy or as a consequence of disease treatment. As neoepitopes originating from retained introns can be presented on the cancer cell surface, the development of personalized cancer vaccines based on IR-derived neoepitopes should be considered. Ultimately, a deeper comprehension about the origins and consequences of aberrant IR may aid in the development of such personalized cancer vaccines

A conserved mammalian mitochondrial isoform of acetyl-CoA carboxylase ACC1 provides the malonyl-CoA essential for mitochondrial biogenesis in tandem with ACSF3

Abstract Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer

In-frame deletion in canine PITRM1 is associated with a severe early-onset epilepsy, mitochondrial dysfunction and neurodegeneration

We investigated the clinical, genetic, and pathological characteristics of a previously unknown severe juvenile brain disorder in several litters of Parson Russel Terriers. The disease started with epileptic seizures at 6-12 weeks of age and progressed rapidly to status epilepticus and death or euthanasia. Histopathological changes at autopsy were restricted to the brain. There was severe acute neuronal degeneration and necrosis diffusely affecting the grey matter throughout the brain with extensive intraneuronal mitochondrial crowding and accumulation of amyloid-beta (A beta). Combined homozygosity mapping and genome sequencing revealed an in-frame 6-bp deletion in the nuclear-encoded pitrilysin metallopeptidase 1 (PITRM1) encoding for a mitochondrial protease involved in mitochondrial targeting sequence processing and degradation. The 6-bp deletion results in the loss of two amino acid residues in the N-terminal part of PITRM1, potentially affecting protein folding and function. Assessment of the mitochondrial function in the affected brain tissue showed a significant deficiency in respiratory chain function. The functional consequences of the mutation were modeled in yeast and showed impaired growth in permissive conditions and an impaired respiration capacity. Loss-of-function variants in human PITRM1 result in a childhood-onset progressive amyloidotic neurological syndrome characterized by spinocerebellar ataxia with behavioral, psychiatric and cognitive abnormalities. Homozygous Pitrm1-knockout mice are embryonic lethal, while heterozygotes show a progressive, neurodegenerative phenotype characterized by impairment in motor coordination and A beta deposits. Our study describes a novel early-onset PITRM1-related neurodegenerative canine brain disorder with mitochondrial dysfunction, A beta accumulation, and lethal epilepsy. The findings highlight the essential role of PITRM1 in neuronal survival and strengthen the connection between mitochondrial dysfunction and neurodegeneration.Peer reviewe

Expression and Evolution of the Non-Canonically Translated Yeast Mitochondrial Acetyl-CoA Carboxylase Hfa1p

<div><p>The <i>Saccharomyces cerevisiae</i> genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the <i>HFA1</i> gene and its evolutionary origin have remained unclear. Here, we determined the <i>HFA1</i> transcription initiation sites by 5′ RACE analysis. Using a novel “Stop codon scanning” approach, we mapped the location of the <i>HFA1</i> translation initiation site to an upstream AUU codon at position −372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. <i>In silico</i> analyses of fungal <i>ACC</i> genes revealed conserved “cryptic” upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δ<i>hfa1</i> baker's yeast mutant phenotype rescue studies using the protoploid <i>Kluyveromyces lactis ACC</i> confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in <i>S. cerevisiae</i> have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5′ upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.</p></div

Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.

<p>All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of <i>HFA1</i>. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to <i>ACC1</i>. The ORFof <i>HFA1</i> annotated in the <i>Saccharomyces</i> Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu <i>et al</i>. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of <i>HFA1</i>.</p

The W1536 8B Δ<i>hfa1</i> strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.

<p>Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δ<i>hfa1</i> or W1536 8B Δ<i>htd2</i> (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 <i>HFA1</i>; −381: YCp33 <i>HFA1</i> −381; −372: YCp33 <i>HFA1</i> −372; −363: YCp33<i>HFA1</i> −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114738#pone-0114738-g002" target="_blank">Fig. 2</a> can be found as supplementary data.</p