New connections between splicing and human disease

doi:10.1016/j.tig.2012.01.001

Trends in Genetics

Volume 28, Issue 4, April 2012, Pages 147-154

https://doi.org/10.1016/j.tig.2012.01.001 Get rights and content

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles termed spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep-sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in particular cancers and pre-cancerous conditions, suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Mis-splicing of crucial genes may underlie the pathologies of all of these diseases. Identifying these genes and understanding the mechanisms involved in their mis-splicing may lead to advancements in diagnosis and treatment.

Section snippets

Intron removal by the spliceosomes

The removal of introns from pre-messenger RNAs by RNA splicing is an essential function in virtually all eukaryotic organisms. In vertebrates, including humans, most genes have multiple introns and most such genes are spliced in more than one pattern to give rise to considerable numbers of alternative mRNA isoforms. These isoforms can have altered protein-coding potential and/or altered regulatory regions and are thought to help generate organism-level complexity from a limited number of genes.

RNA splicing and disease

The fact that genes are composed of multiple segments has a variety of functional consequences, some positive and some negative. On the positive side, it is clear that the vast majority of mammalian genes are spliced and otherwise processed in different ways, thereby allowing the expression of many different mRNA isoforms and protein products from a relatively small number of nuclear genes [20]. This diversity of gene products is central to the complex processes of growth, development and

Mutations in a spliceosomal RNA cause disease

Microcephalic osteodysplastic primordial dwarfism type I (MOPD I, also known as Taybi–Linder Syndrome) is an autosomal recessive genetic disorder that is rare in most populations but is fairly common among the Amish in Ohio [28]. This condition is diagnosed on the basis of characteristic neurological and skeletal abnormalities as well as slow intra-uterine growth and greatly reduced size at term. Lifespans of MOPD I patients are somewhat variable – from only a few months to close to 13 years

Current questions raised by the MOPD I mutations

Among the immediate questions raised by these findings are: first, what is the molecular defect in the mutant U4atac snRNAs? – in other words at what point in the assembly of the snRNP complexes or their function in splicing do they fail? This problem could be investigated using in vitro biochemical studies of NHP2L1 and PRP31 protein binding to the mutant snRNA to assay for defects in direct binding. Because MOPD I patient-derived cell lines are available, the ability of the mutant U4atac

Splicing-factor mutations in myeloid neoplasms and leukemias

All the above examples are linked to inherited mutations in splicing components. Recently, a striking example of the effects of acquired somatic mutations in splicing factors has been described. The sequencing of the DNA from abnormal blood cells from patients with several types of leukemia and pre-leukemic syndromes has shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins 44, 45, 46, 47. These diseases include myelodysplastic syndrome

Concluding remarks

These recent results emphasize the role of mutations in the essential machinery of pre-mRNA splicing in the realm of human health and disease. Although the minor U12-dependent spliceosome has been relatively poorly studied, it is now clear that defects in this machine can have substantial effects on growth and development. Future studies that identify the target genes have the potential to uncover important pathways in the various cell types affected in MOPD I. Furthermore, the linkage of

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The objective of this chapter is to provide an overview of the chemical and mechanistic classes of oligonucleotide-based therapeutics (ONTs), to describe the general strategies used in developing the nonclinical safety program for this evolving class of molecules, and to provide a general characterization of the class- and chemistry-dependent toxicity observed in species used for nonclinical safety assessment. The most well-described pharmacologic mechanism for synthetic ONTs include the antisense targeting of specific RNA transcripts, although numerous others exist (e.g., modulation of pre-mRNA splicing, aptameric targeting of proteins, inhibition of mRNA translation, exon skipping/inclusion and modulation of noncoding RNA). Most ONTs advanced to clinical testing have been unconjugated and formulated in simple aqueous solutions. For such “naked” ONTs, the physicochemical and pharmacokinetic properties (e.g., length, solubility, charge-to-mass ratio, hydrophilicity, tissue distribution, metabolism, and protein binding, among others) are generally comparable for molecules within each chemical class. Thus, the toxicologic properties of naked ONTs are qualitatively similar within a chemical subclass, as a whole. Targeted delivery of ONTs facilitated by the use of complex formulations and/or conjugation to ligands has much promise and is dominating the class of ONTs being tested currently. Target delivery platforms are likely going to impact the physiochemical properties of ONTs, and what is known about naked ONTs may not fully represent the characteristics of these more complex molecules. Nonetheless, the preclinical and clinical experience gained with various classes of ONTs is extensive, and this therapeutic modality has matured over the last 20–30 years, with 10 approved medicines across numerous therapeutic indications.
Role of alternative splicing in health and diseases
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Alternative splicing (AS) is a characteristic of many genes, in which several different transcripts can be originated from the same primary mRNA. These variants are called transcripts or isoforms. AS provides the required diversity to apply gene products in different conditions or tissues. More than 90% of human intron-containing genes undergo AS. Furthermore, these genes are thought to encode two or more isoforms, which are diversified across tissues and developmental stages. Each biological function role of our body like cell death, controlling cell division, control of gene expression is played by AS. Different heritable diseases are either related to irregular/abnormal AS or resulted from irregular/abnormal AS. And about half of the genetic disorders which are inherited are related to SNPs that are also the result of wreckage of splicing events. Profiling normal and disease tissues shows significant changes in AS related to the progression of disease. Genome-wide platforms for assessing AS are a powerful tool for understanding the pathological role of this process. Abnormal AS has been related to several diseases, including cancer. AS is a potential target for therapeutics, and there is active research on finding new biomarkers. Finding disease specific biomarkers might allow; early diagnosis of diseases and their prevention strategies to assure healthy longevity, and identification of personalized medicine. This chapter lightens about basic of gene expression, regulation via AS, regulators of AS, technique to assess the AS, brief overview of various algorithms for finding AS, and role of AS in disease.
An intron mutation of HNF1A causes abnormal splicing and impairs its activity as a transcription factor
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Mutations in HNF1A are associated with Maturity Onset Diabetes of the Young type 3 (MODY3) and most of them are in the coding region. Herein, we identified an intron mutation at the 6th nucleotide upstream of the end of intron 7 of HNF1A, named IVS7-6G > A, in a patient with early-onset diabetes. The “minigene” assay showed that IVS7-6G > A produced two aberrant mRNA variants translating into two truncated proteins: L502S fs* and G437A fs*, both affecting HNF1A transactivation domain (TAD). To determine functional consequences of IVS7-6G > A mutation, we made plasmids encoding truncated HNF1A containing different portions of HNF1A TAD and found that the TAD of HNF1A is important not only for its regulatory activities, but also for its nuclearization, and the residues 282–501 was more essential than 502–631. Our data suggested IVS7-6G > A impaired HNF1A splicing and may contribute to the pathogenesis of MODY3.
Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms
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The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.
Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency
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We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency.
We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain.
We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke’s pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype.
Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.
Impact of alternative splicing on mechanisms of resistance to anticancer drugs
2021, Biochemical Pharmacology
Citation Excerpt :
The degree of complexity due to AS of pre-mRNA in target genes is increased because components of the splicing machinery and regulatory proteins involved in exon-recognition can themselves undergo AS, generating different isoforms (Table 1), which can modify the overall result of the splicing process. Since splicing dysregulation is a hallmark of cancer, it is not surprising that there are more than 15,000 tumor-associated SVs described in a wide variety of malignancies [40,41]. For instance, in a cell model of breast cancer, 1723 splicing alterations in target genes and changes in the expression of 41 splicing factors have been reported [42].
A shared characteristic of many tumors is the lack of response to anticancer drugs. Multiple mechanisms of pharmacoresistance (MPRs) are involved in permitting cancer cells to overcome the effect of these agents. Pharmacoresistance can be primary (intrinsic) or secondary (acquired), i.e., triggered or enhanced in response to the treatment. Moreover, MPRs usually result in the lack of sensitivity to several agents, which accounts for diverse multidrug-resistant (MDR) phenotypes. MPRs are based on the dynamic expression of more than one hundred genes, constituting the so-called resistome. Alternative splicing (AS) during pre-mRNA maturation results in changes affecting proteins involved in the resistome. The resulting splicing variants (SVs) reduce the efficacy of anticancer drugs by lowering the intracellular levels of active agents, altering molecular targets, enhancing both DNA repair ability and defensive mechanism of tumors, inducing changes in the balance between pro-survival and pro-apoptosis signals, modifying interactions with the tumor microenvironment, and favoring malignant phenotypic transitions. Reasons accounting for cancer-associated aberrant splicing include mutations that create or disrupt splicing sites or splicing enhancers or silencers, abnormal expression of splicing factors, and impaired signaling pathways affecting the activity of the splicing machinery. Here we have reviewed the impact of AS on MPR in cancer cells.

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Trends in Genetics

ReviewNew connections between splicing and human disease

Section snippets

Intron removal by the spliceosomes

RNA splicing and disease

Mutations in a spliceosomal RNA cause disease

Current questions raised by the MOPD I mutations

Splicing-factor mutations in myeloid neoplasms and leukemias

Concluding remarks

Cell

J. Mol. Biol.

Cell

Mol. Cell

Cell

Mol. Cell

Cell

Cell

FEBS Lett.

J. Biol. Chem.

Spliced segments at the 5′ terminus of adenovirus 2 late mRNA

Proc. Natl. Acad. Sci. U.S.A.

A reappraisal of non-consensus mRNA splice sites

Nucl. Acids Res.

Requirement of U12 snRNA for the in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns

Science

An early evolutionary origin for the minor spliceosome

Nature

U12 type introns were lost at multiple occasions during evolution

BMC Genomics

U12DB: a database of orthologous U12-type spliceosomal introns

Nucleic Acids Res.

A computational scan for U12-dependent introns in the human genome sequence

Nucleic Acids Res.

Spliceosome structure and function

The spliceosomal proteome: at the heart of the largest cellular ribonucleoprotein machine

Proteomics

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

Mol. Cell. Biol.

Splicing of a rare class of introns by the U12-dependent spliceosome

Biol. Chem.

Identification of both shared and distinct proteins in the major and minor spliceosomes

Science

SR proteins and related factors in alternative splicing

Adv. Exp. Med. Biol.

Alternative isoform regulation in human tissue transcriptomes

Nature

The pathobiology of splicing

J. Pathol.

Splicing in disease: disruption of the splicing code and the decoding machinery

Nat. Rev. Genet.

Review
New connections between splicing and human disease