Exitron

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Exitrons (exonic introns) are produced through alternative splicing and have characteristics of both introns and exons, but are described as retained introns. Even though they are considered introns, which are typically cut out of pre mRNA sequences, there are significant problems that arise when exitrons are spliced out of these strands, with the most obvious result being altered protein structures and functions. They were first discovered in plants, but have recently been found in metazoan species as well.

Contents

Alternative splicing

Exitrons are a result of alternative splicing (AS), in which introns are typically cut out of a pre mRNA sequence, while exons remain in the sequence and are translated into proteins. The same sequence within a pre mRNA strand can be considered an intron or exon depending on the desired protein to be produced. As a result, different final mRNA sequences are generated and a large variety of proteins can be made from one single gene. [1] Mutations that exist in these sequences can also alter the way in which a sequence is spliced and as a result, change the protein produced. [2] Splicing mutations of a mRNA sequence has been found to account for 15-60% of human genetic diseases, which suggests there may be a crucial role of exitrons in organ homeostasis. [3] [4]

Discovery

A previous study had looked at alternative splicing in Rockcress (Arabidopsis) plants and pinpointed characteristics of retained introns in sequences. They had a subset of what they called "cryptic introns" that did not contain stop codons and are now deemed exitrons. [5] The same researchers conducted further studies on their newly discovered exitrons and found 1002 exitrons in 892 Rockcress genes, a flowering plant that has been used to model exitrons. [4] Although they were discovered in plants, exitrons have also been found in other metazoan species and humans as well. [4] [6] A recent comprehensive analysis of exitron splicing in 33 cancer types highlighted the abundance and impact of exitrons in human cancers. [7] This study revealed exitron splicing disrupts functional protein domains, causing cancer driver effects and introducing a new potential source of neoantigens. [7] [8]

Distinguishing these regions from typical introns

Transcripts with exitrons in their sequences can be distinguished from those with retained introns in several ways: (1) transcripts containing exitrons are transported out of the nucleus to be translated, whereas those containing introns are identified as incompletely processed and are kept in the nucleus where they cannot be translated. (2) only transcripts with exitrons of lengths not divisible by three have the potential to incorporate premature termination sequences, while sequences with introns normally result in premature termination. Thus, frameshifting exitron events were more likely to evade nonsense-mediated decay (NMD) than intron retentions. [7] (3) exitron transcripts are usually the major isoforms, but those with introns are only present in small amounts. [6] (4) exitrons had distinct cis-acting features such as weak 5′ and 3′ splice sites, high GC content, and short length compared to retained introns. [7]

Characteristics

Exitrons are considered introns, but have characteristics of both introns and exons. They originated from ancestral coding exons, but have weaker splice site signals than other introns. Exitrons have been found to be longer and have a higher GC content than intron regions and constitutive introns. However, they are of similar size to constitutive exons and their GC content is lower compared to other exons. [4] Exitrons lack stop codons within their sequences, have synonymous substitutions, and are most commonly found in multiples of three nucleotides. [6] Exitron sequences contain sites for numerous post-translational modifications, including sumoylation, ubiquitylation, S-nitrosylation, and lysine acetylation. The ability of exitron splicing (EIS) to alter protein states demonstrates the effect it can have on proteome assortment. [4]

In Arabidopsis

Exitron splicing affects 3.3% of Arabidopsis protein coding genes. 11% of intron regions were composed of exitrons and 3.7% of AS events detected in a sample were exitron splicings. The regulation of EIS in tissues is controlled by certain stresses, which serves as a regulatory role in plant adaptation and development. [4]

In human cancers

A analysis showed that exitron splicing affected 63% of human coding genes and that 95% of those events were tumor-specific. [7] It was found that exitron splicing occurred more frequently in cancer tissues (63%), compared to normal human tissue cells (17%), with the highest rate of exitron splicing occurring in ovarian, esophageal, stomach, and acute myeloid leukemia tumors. [7] Using a generalized additive model, researchers determined that exitron splicing dysregulation in cancers could largely be explained by differential expression of splicing factors. [7]

Effects

Exitron splicing has been found to be a conserved strategy for increasing proteome plasticity in both plants and animals since it affects plant and human protein features in a similar manner. [4] When exitrons are spliced out of a sequence, it has resulted in internally deleted proteins and affected protein domains, disordered regions, and various post-translational modification sites that impact protein function. [6] Spliced exitrons can result in premature termination of a protein, while in contrast, a non-spliced exitron results in a full-length protein. [4]

The processing of these exitrons has been found to be sensitive to cell types and environmental conditions and their splicing is linked to cancer. [4] [6] [9] The impairment of EIS can potentially contribute to the initiation of cancer formation through its effect on several cancer-related genes. These genes include oncogenes and genes involved in cell adhesion, migration, and metastasis. [4]

EIS also facilitated the discovery of novel cancer driver genes. One of the significantly exitron-spliced genes (SEGs), NEFH, which rarely experiences mutations, was identified as a novel tumor suppressor in prostate cancer. Exitron splicing has the potential to introduce highly immunogenic neoantigens, which can be targetable with immunotherapy, thereby providing a promising avenue for cancer treatment. [7]

See also

Related Research Articles

<span class="mw-page-title-main">Exon</span> A region of a transcribed gene present in the final functional mRNA molecule

An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature RNA. Just as the entire set of genes for a species constitutes the genome, the entire set of exons constitutes the exome.

An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word intron is derived from the term intragenic region, i.e. a region inside a gene. The term intron refers to both the DNA sequence within a gene and the corresponding RNA sequence in RNA transcripts. The non-intron sequences that become joined by this RNA processing to form the mature RNA are called exons.

<span class="mw-page-title-main">RNA splicing</span> Process in molecular biology

RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns and splicing back together exons. For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). There exist self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule. The process of transcription, splicing and translation is called gene expression, the central dogma of molecular biology.

<span class="mw-page-title-main">Alternative splicing</span> Process by which a gene can code for multiple proteins

Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. This means the exons are joined in different combinations, leading to different (alternative) mRNA strands. Consequently, the proteins translated from alternatively spliced mRNAs usually contain differences in their amino acid sequence and, often, in their biological functions.

<span class="mw-page-title-main">Protein isoform</span> Forms of a protein produced from different genes

A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have unique functions. A set of protein isoforms may be formed from alternative splicings, variable promoter usage, or other post-transcriptional modifications of a single gene; post-translational modifications are generally not considered. Through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein.

Trans-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated. It is usually found in eukaryotes and mediated by the spliceosome, although some bacteria and archaea also have "half-genes" for tRNAs.

<span class="mw-page-title-main">SR protein</span>

SR proteins are a conserved family of proteins involved in RNA splicing. SR proteins are named because they contain a protein domain with long repeats of serine and arginine amino acid residues, whose standard abbreviations are "S" and "R" respectively. SR proteins are ~200-600 amino acids in length and composed of two domains, the RNA recognition motif (RRM) region and the RS domain. SR proteins are more commonly found in the nucleus than the cytoplasm, but several SR proteins are known to shuttle between the nucleus and the cytoplasm.

<span class="mw-page-title-main">Primary transcript</span> RNA produced by transcription

A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNAs are modified in preparation for translation. For example, a precursor mRNA (pre-mRNA) is a type of primary transcript that becomes a messenger RNA (mRNA) after processing.

RNA-binding proteins are proteins that bind to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes. RBPs contain various structural motifs, such as RNA recognition motif (RRM), dsRNA binding domain, zinc finger and others. They are cytoplasmic and nuclear proteins. However, since most mature RNA is exported from the nucleus relatively quickly, most RBPs in the nucleus exist as complexes of protein and pre-mRNA called heterogeneous ribonucleoprotein particles (hnRNPs). RBPs have crucial roles in various cellular processes such as: cellular function, transport and localization. They especially play a major role in post-transcriptional control of RNAs, such as: splicing, polyadenylation, mRNA stabilization, mRNA localization and translation. Eukaryotic cells express diverse RBPs with unique RNA-binding activity and protein–protein interaction. According to the Eukaryotic RBP Database (EuRBPDB), there are 2961 genes encoding RBPs in humans. During evolution, the diversity of RBPs greatly increased with the increase in the number of introns. Diversity enabled eukaryotic cells to utilize RNA exons in various arrangements, giving rise to a unique RNP (ribonucleoprotein) for each RNA. Although RBPs have a crucial role in post-transcriptional regulation in gene expression, relatively few RBPs have been studied systematically.It has now become clear that RNA–RBP interactions play important roles in many biological processes among organisms.

Exon shuffling is a molecular mechanism for the formation of new genes. It is a process through which two or more exons from different genes can be brought together ectopically, or the same exon can be duplicated, to create a new exon-intron structure. There are different mechanisms through which exon shuffling occurs: transposon mediated exon shuffling, crossover during sexual recombination of parental genomes and illegitimate recombination.

<span class="mw-page-title-main">Splice site mutation</span> Mutation at a location where intron splicing takes place

A splice site mutation is a genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing takes place during the processing of precursor messenger RNA into mature messenger RNA. Splice site consensus sequences that drive exon recognition are located at the very termini of introns. The deletion of the splicing site results in one or more introns remaining in mature mRNA and may lead to the production of abnormal proteins. When a splice site mutation occurs, the mRNA transcript possesses information from these introns that normally should not be included. Introns are supposed to be removed, while the exons are expressed.

An interrupted gene is a gene that contains expressed regions of DNA called exons, split with unexpressed regions called introns. Exons provide instructions for coding proteins, which create mRNA necessary for the synthesis of proteins. Introns are removed by recognition of the donor site and the splice acceptor site. The architecture of the interrupted gene allows for the process of alternative splicing, where various mRNA products can be produced from a single gene. The function of introns are still not fully understood and are called noncoding or junk DNA.

An exonic splicing silencer (ESS) is a short region of an exon and is a cis-regulatory element. A set of 103 hexanucleotides known as FAS-hex3 has been shown to be abundant in ESS regions. ESSs inhibit or silence splicing of the pre-mRNA and contribute to constitutive and alternate splicing. To elicit the silencing affect, ESSs recruit proteins that will negatively affect the core splicing machinery.

<span class="mw-page-title-main">RBM9</span> Protein-coding gene in the species Homo sapiens

RNA binding motif protein 9 (RBM9), also known as Rbfox2, is a protein which in humans is encoded by the RBM9 gene.

Post-transcriptional regulation is the control of gene expression at the RNA level. It occurs once the RNA polymerase has been attached to the gene's promoter and is synthesizing the nucleotide sequence. Therefore, as the name indicates, it occurs between the transcription phase and the translation phase of gene expression. These controls are critical for the regulation of many genes across human tissues. It also plays a big role in cell physiology, being implicated in pathologies such as cancer and neurodegenerative diseases.

Periannan Senapathy is a molecular biologist, geneticist, author and entrepreneur. He is the founder, president and chief scientific officer at Genome International Corporation, a biotechnology, bioinformatics, and information technology firm based in Madison, Wisconsin, which develops computational genomics applications of next-generation DNA sequencing (NGS) and clinical decision support systems for analyzing patient genome data that aids in diagnosis and treatment of diseases.

Chimeric RNA, sometimes referred to as a fusion transcript, is composed of exons from two or more different genes that have the potential to encode novel proteins. These mRNAs are different from those produced by conventional splicing as they are produced by two or more gene loci.

<span class="mw-page-title-main">Circular RNA</span> Type of RNA found in cells

Circular RNA is a type of single-stranded RNA which, unlike linear RNA, forms a covalently closed continuous loop. In circular RNA, the 3' and 5' ends normally present in an RNA molecule have been joined together. This feature confers numerous properties to circular RNA, many of which have only recently been identified.

<span class="mw-page-title-main">Minigene</span>

A minigene is a minimal gene fragment that includes an exon and the control regions necessary for the gene to express itself in the same way as a wild type gene fragment. This is a minigene in its most basic sense. More complex minigenes can be constructed containing multiple exons and intron(s). Minigenes provide a valuable tool for researchers evaluating splicing patterns both in vivo and in vitro biochemically assessed experiments. Specifically, minigenes are used as splice reporter vectors and act as a probe to determine which factors are important in splicing outcomes. They can be constructed to test the way both cis-regulatory elements and trans-regulatory elements affect gene expression.

The split gene theory is a theory of the origin of introns, long non-coding sequences in eukaryotic genes between the exons. The theory holds that the randomness of primordial DNA sequences would only permit small (< 600bp) open reading frames (ORFs), and that important intron structures and regulatory sequences are derived from stop codons. In this introns-first framework, the spliceosomal machinery and the nucleus evolved due to the necessity to join these ORFs into larger proteins, and that intronless bacterial genes are less ancestral than the split eukaryotic genes. The theory originated with Periannan Senapathy.

References

  1. ↑ 1. Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., Walter, P. Molecular Biology of the Cell. 6. New York: Garland Science; 2015. p. 319-320, 415.
  2. ↑ 2. Edwalds-Gilbert, G. Regulation of mRNA Splicing by Signal Transduction. [Internet]. Scitable.; [cited 2016 Feb 15]. Available from http://www.nature.com/scitable/topicpage/regulation-of-mrna-splicing-by-signal-transduction-14128469
  3. ↑ 3. Wang, G. S., Cooper, T. A. Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet. 2007;8(10): 749-761.
  4. 1 2 3 4 5 6 7 8 9 10 4.Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria (July 2015). "Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity". Genome Research. 25 (7): 995–1007. doi:10.1101/gr.186585.114. ISSN   1088-9051. PMC   4484396 . PMID   25934563.
  5. ↑ Marquez, Yamile; Brown, John W.S.; Simpson, Craig; Barta, Andrea; Kalyna, Maria (June 2012). "Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis". Genome Research. 22 (6): 1184–1195. doi: 10.1101/gr.134106.111 .
  6. 1 2 3 4 5 5. Staiger, D., Simpson, G. G. Enter exitrons. [Internet]. BioMed Central.; [cited 2016 Feb 15]. Available from http://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0704-3
  7. 1 2 3 4 5 6 7 8 Wang, Ting-You; Liu, Qi; Ren, Yanan; Alam, Sk. Kayum; Wang, Li; Zhu, Zhu; Hoeppner, Luke H.; Dehm, Scott M.; Cao, Qi; Yang, Rendong (May 2021). "A pan-cancer transcriptome analysis of exitron splicing identifies novel cancer driver genes and neoepitopes". Molecular Cell. 81 (10): 2246–2260. doi: 10.1016/j.molcel.2021.03.028 .
  8. ↑ Sellars, MacLean C.; Wu, Catherine J.; Fritsch, Edward F. (2022-07-21). "Cancer vaccines: Building a bridge over troubled waters". Cell. 185 (15): 2770–2788. doi:10.1016/j.cell.2022.06.035.
  9. ↑ 6. MEMBS E-News. Exitron Splicing: New Aspect of Gene Regulation. [Internet]. Middle East Molecular Biology Society.; [cited 2016 Feb 15]. Available from http://enews.membs.org/Exitron-Splicing--New-Aspect-of-Gene-Regulation Archived 2016-05-08 at the Wayback Machine
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