Minigene

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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. [1] [2] Specifically, minigenes are used as splice reporter vectors (also called exon-trapping 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 (RNA effects) and trans-regulatory elements (associated proteins/splicing factors) affect gene expression. [3]

Contents

pre-mRNA splice patterns showing constitutive exons and introns and the inserted fragment. Orange lines show alternative splicing outcomes as dictated by the exonic sequences and intronic sequences (yellow and green bands) that influence splicing. These sequences may be splicing enhancers or silencers, polypyrimidine tract binding protein sites or other elements. Splice2.JPG
pre-mRNA splice patterns showing constitutive exons and introns and the inserted fragment. Orange lines show alternative splicing outcomes as dictated by the exonic sequences and intronic sequences (yellow and green bands) that influence splicing. These sequences may be splicing enhancers or silencers, polypyrimidine tract binding protein sites or other elements.

History

Minigenes were first described as the somatic assembly of DNA segments and consisted of DNA regions known to encode the protein and the flanking regions required to express the protein. The term was first used in a paper in 1977 to describe the cloning of two minigenes that were designed to express a peptide. [4]

RNA splicing was discovered in the late 1970s through the study of adenoviruses that invade mammals and replicate inside them. Researchers identified RNA molecules that contained sequences from noncontiguous parts of the virus’s genome. This discovery led to the conclusion that regulatory mechanisms existed which affected mature RNA and the genes it expresses. [5] Using minigenes as a splice reporting vector to explore the effects of RNA splicing regulation naturally followed and remains the major use of minigenes to date.

Types

In order to provide a good minigene model, the gene fragment should have all of the necessary elements to ensure it exhibits the same alternative splicing (AS) patterns as the wild type gene, i.e., the length of the fragment must include all upstream and downstream sequences which can affect its splicing. [1] [2] Therefore, most minigene designs begin with a thorough in silico analysis of the requirements of the experiment before any "wet" lab work is conducted. [6] With the advent of Bioinformatics and widespread use of computers, several good programs now exist for the identification of cis-acting control regions that affect the splicing outcomes of a gene [7] [8] and advanced programs can even consider splicing outcomes in various tissue types. [9] Differences in minigenes are usually reflected in the final size of the fragment, which is in turn a reflection of the complexity of the minigene itself. The number of foreign DNA elements (exon and introns) inserted into the constitutive exons and introns of a given fragment varies with the type of experiment and the information being sought. A typical experiment might involve wild type minigenes which are expected to express genes normally in a comparison run against genetically engineered allelic variations which replace the wild-type gene and have been cloned into the same flanking sequences as the original fragment. These types of experiments help to determine the effect of various mutations on pre-mRNA splicing. [3]

Construction

Once a suitable genomic fragment is chosen (Step 1), the exons and introns of the fragment can be inserted and amplified, along with the flanking constitutive exons and introns of the original gene, by PCR. The primers for PCR can be chosen so that they leave "sticky ends" at 3' sense and anti-sense strands (Step 2). These "sticky-ends" can be easily incorporated into a TOPO Vector by ligation into a commercially available source which has ligase already attached to it at the sight of incorporation [10] (Step 3). The subsequent TOPO Vectors can be transfected into E.coli cells (Step 4). After incubation, total RNA can be extracted from the bacterial colonies and analyzed using RT-PCR to quantify ratios of exon inclusion/exclusion (step 5). The minigene can be transfected into different cell types with various splicing factors to test trans-acting elements (Step 6). The expressed genes or the proteins they encode can be analyzed to evaluate splicing components and their effects via a variety of methods including hybridization or size-exclusion chromatography. [1] [2]

A typical cycle for constructing a minigene. Minigen cycle1.JPG
A typical cycle for constructing a minigene.

Uses

RNA splicing errors have been estimated to occur in a third of genetic diseases.[ citation needed ] To understand pathogenesis and identify potential targets of therapeutic intervention in these diseases, explicating the splicing elements involved is essential. [11] Determining the complete set of components involved in splicing presents many challenges due to the abundance of alternative splicing, which occurs in most human genes, and the specificity in which splicing is carried out in vivo. [2] Splicing is distinctly conducted from cell type to cell type and across different stages of cellular development. Therefore, it is critical that any in vitro or bioinformatic assumptions about splicing regulation are confirmed in vivo. [12] Minigenes are used to elucidate cis-regulatory elements, trans-regulatory elements and other regulators of pre-mature RNA splicing in vivo. [2] Minigenes have been applied to the study of a diverse array of genetic diseases due to the aforementioned abundance of alternatively spliced genes and the specificity and variation observed in splicing regulation. [1] [2] [12] The following are examples of minigene use in various diseases. While it is not an exhaustive list, it does provide a better understanding of how minigenes are utilized.

Endocrine diseases

RNA splicing errors can have drastic effects on how proteins function, including the hormones secreted by the endocrine system. These effects on hormones have been identified as the cause of many endocrine disorders including thyroid-related pathological conditions, rickets, hyperinsulinemic hypoglycemia and congenital adrenal hyperplasia. [13] One specific example of a splicing error causing an endocrine disease that has been studied using minigenes is a type of growth hormone deficiency called isolated growth hormone deficiency (IGHD), a disease that results in growth failure. IGHD type II is an autosomal dominant form caused by a mutation in the intervening sequence (IVS) adjacent to exon 3 of the gene encoding growth hormone 1, the GH-1 gene. This mutated form of IVS3 causes exon 3 to be skipped in the mRNA product. The mRNA (-E3) encodes a truncated form of hGH that then inhibits normal hGH secretion. Minigenes were used to determine that a point mutation within an intron splice enhancer (ISE) embedded in IVS3 was to blame for the skipping of E3. Moreover, it was determined that the function of the ISE is influenced by a nearby transposable AC element, revealing that this particular splicing error is caused by a trans-acting factor. [14]

Neurodegenerative diseases

Accumulation of tau protein is associated with neurodegenerative diseases including Alzheimer's and Parkinson's diseases as well as other tauopathies. [15] Tau protein isoforms are created by alternative splicing of exons 2, 3 and 10. The regulation of tau splicing is specific to stage of development, physiology and location. Errors in tau splicing can occur in both exons and introns and, depending on the error, result in changes to protein structure or loss of function. [16] Aggregation of these abnormal tau proteins correlates directly with pathogenesis and disease progression. Minigenes have been used by several researchers to help understand the regulatory components responsible for mRNA splicing of the TAU gene. [15] [16] [17]

Cancer

Cancer is a complex, heterogeneous disease that can be hereditary or the result of environmental stimuli. [18] Minigenes are used to help oncologists understand the roles pre-mRNA splicing plays in different cancer types. Of particular interest are cancer specific genetic mutations that disrupt normal splicing events, including those affecting spliceosome components and RNA-binding proteins such as heterogeneous nuclear ribonucleoparticules (hnRNP), serine/arginine-rich (SR) proteins and small ribonucleoproteins (snRNP). [19] [20] Proteins encoded by aberrantly spliced pre-mRNAs are functionally different and contribute to the characteristic anomalies exhibited by cancer cells, including their ability to proliferate, invade and undergo angiogenesis, and metastasis. [20] Minigenes help researchers identify genetic mutations in cancer that result in splicing errors and determine the downstream effects those splicing errors have on gene expression. [21] Using knowledge obtained from studies employing minigenes, oncologists have proposed tests designed to detect products of abnormal gene expression for diagnostic purposes. [22] Additionally, the prospect of using minigenes as a cancer immunotherapy is being explored. [23] [24]

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.

The coding region of a gene, also known as the coding sequence(CDS), is the portion of a gene's DNA or RNA that codes for protein. Studying the length, composition, regulation, splicing, structures, and functions of coding regions compared to non-coding regions over different species and time periods can provide a significant amount of important information regarding gene organization and evolution of prokaryotes and eukaryotes. This can further assist in mapping the human genome and developing gene therapy.

<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.

In computational biology, gene prediction or gene finding refers to the process of identifying the regions of genomic DNA that encode genes. This includes protein-coding genes as well as RNA genes, but may also include prediction of other functional elements such as regulatory regions. Gene finding is one of the first and most important steps in understanding the genome of a species once it has been sequenced.

<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.

<span class="mw-page-title-main">Nonsense-mediated decay</span> Elimination of mRNA with premature stop codons in eukaryotes

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that exists in all eukaryotes. Its main function is to reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs could, in some cases, lead to deleterious gain-of-function or dominant-negative activity of the resulting proteins.

<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.

In molecular biology, an exonic splicing enhancer (ESE) is a DNA sequence motif consisting of 6 bases within an exon that directs, or enhances, accurate splicing of heterogeneous nuclear RNA (hnRNA) or pre-mRNA into messenger RNA (mRNA).

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 effect, ESSs recruit proteins that will negatively affect the core splicing machinery.

<span class="mw-page-title-main">TIA1</span> Mammalian protein found in Homo sapiens

TIA1 or Tia1 cytotoxic granule-associated rna binding protein is a 3'UTR mRNA binding protein that can bind the 5'TOP sequence of 5'TOP mRNAs. It is associated with programmed cell death (apoptosis) and regulates alternative splicing of the gene encoding the Fas receptor, an apoptosis-promoting protein. Under stress conditions, TIA1 localizes to cellular RNA-protein conglomerations called stress granules. It is encoded by the TIA1 gene.

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.

<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.

Exitrons 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.

<span class="mw-page-title-main">Shapiro Senapathy algorithm</span>

The Shapiro Senapathy algorithm (S&S) is an algorithm for predicting splice junctions in genes of animals and plants. This algorithm has been used to discover disease-causing splice site mutations and cryptic splice sites.

ZTTK syndrome is a rare disease caused in humans by a genetic mutation of the SON gene. Common symptoms include developmental delay and sometimes moderate to several intellectual disability.

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

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  2. 1 2 3 4 5 6 Cooper, Thomas A. (December 2005). "Use of minigene systems to dissect alternative splicing elements". Methods. 37 (4): 331–340. doi:10.1016/j.ymeth.2005.07.015. PMID   16314262.
  3. 1 2 Desviat, LR; Pérez, B; Ugarte, M (2012). "Minigenes to Confirm Exon Skipping Mutations". Exon Skipping. Methods Mol. Biol. Vol. 867. pp. 37–47. doi:10.1007/978-1-61779-767-5_3. ISBN   978-1-61779-766-8. PMID   22454053.
  4. ↑ Poonian, MS; McComas, WW; Nussbaum, AL (1977). "Chemical synthesis of two deoxyribododecanucleotides for the attachment of restriction termini to an artificial minigene". Gene. 1 (5–6): 357–72. doi:10.1016/0378-1119(77)90040-3. PMID   590743.
  5. ↑ Clancy, S (2008). "RNA splicing: introns, exons and spliceosome". Nature Education. 1 (31).
  6. ↑ Burge, Christopher. "Burge Lab Software" . Retrieved 7 May 2014.
  7. ↑ Divina, Petr; Kvitkovicova, Andrea; Buratti, Emanuele; Vorechovsky, Igor (14 January 2009). "Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping". European Journal of Human Genetics. 17 (6): 759–765. doi:10.1038/ejhg.2008.257. PMC   2947103 . PMID   19142208.
  8. ↑ Grodecká, Lucie; Lockerová, Pavla; Ravčuková, Barbora; Buratti, Emanuele; Baralle, Francisco E.; Dušek, Ladislav; Freiberger, Tomáš; Spilianakis, Charalampos Babis (21 February 2014). "Exon First Nucleotide Mutations in Splicing: Evaluation of In Silico Prediction Tools". PLOS ONE. 9 (2): e89570. Bibcode:2014PLoSO...989570G. doi: 10.1371/journal.pone.0089570 . PMC   3931810 . PMID   24586880.
  9. ↑ Barash, Yoseph; Vaquero-Garcia, Jorge; González-Vallinas, Juan; Xiong, Hui; Gao, Weijun; Lee, Leo J.; Frey, Brendan J. (2013). "AVISPA: a web tool for the prediction and analysis of alternative splicing". Genome Biology. 14 (10): R114. doi: 10.1186/gb-2013-14-10-r114 . PMC   4014802 . PMID   24156756.
  10. ↑ "Steps in producing a TOPO Vector". Life sciences. Retrieved 7 May 2014.
  11. ↑ Lim, Kian; Huat; Ferraris, Luciana; Filloux, Madeleine E.; Raphael, Benjamin J.; Fairbrother, William G. (2011). "Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes". Proceedings of the National Academy of Sciences. 108 (27): 11093–6. Bibcode:2011PNAS..10811093H. doi: 10.1073/pnas.1101135108 . PMC   3131313 . PMID   21685335.
  12. 1 2 Stamm, Stefan. "Stamms-lab.net". Archived from the original on 9 December 2013. Retrieved 26 March 2014.
  13. ↑ Rosaria de Miranda, Elizabete (2009). "Splicing variants impact in thyroid normal physiology and pathological conditions". Arq Bras Endocrinol Metabol. 53 (6): 709–714. doi: 10.1590/S0004-27302009000600003 . PMID   19893912.
  14. ↑ Mullis, PE (2010). "Genetics of isolated growth hormone deficiency". J Clin Res Pediatr Endocrinol. 2 (2): 52–62. doi:10.4274/jcrpe.v2i2.52. PMC   3014602 . PMID   21274339.
  15. 1 2 Kar, Amar; Fushimi, Kazuo; Zhou, Xiaohong; Ray, Payal; Shi, Chen; Chen, Xiaoping; Liu, Zhiren; Chen, She; Wu, Jane Y. (2011). "RNA Helicase p68 (DDX5) Regulates tau Exon 10 Splicing by Modulating a Stem-Loop Structure at the 5′ Splice Site". Mol. Cell. Biol. 31 (9): 1812–1821. doi:10.1128/MCB.01149-10. PMC   3133221 . PMID   21343338.
  16. 1 2 Rodriguez-Martin, Teresa; Karen Anthony; Mariano A. Garcia-Blanco; S. Gary Mansfield; Brian H. Anderton; Jean-Marc Gallo (2009). "Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing". Hum Mol Genet. 18 (17): 3266–3273. doi:10.1093/hmg/ddp264. PMC   2722988 . PMID   19498037.
  17. ↑ Anfossi, M; Vuono, R; Maletta, R; Virdee, K; Mirabelli, M; Colao, R; Puccio, G; Bernardi, L; Frangipane, F; Gallo, M; Geracitano, S; Tomaino, C; Curcio, SA; Zannino, G; Lamenza, F; Duyckaerts, C; Spillantini, MG; Losso, MA; Bruni, AC (2011). "Compound heterozygosity of 2 novel MAPT mutations in frontotemporal dementia". Neurobiol Aging. 32 (4): 757.e1–757.e11. doi:10.1016/j.neurobiolaging.2010.12.013. PMID   21295377. S2CID   5176440.
  18. ↑ Rajan, P.; Elliott, DJ; Robson, CN; Leung, HY (Aug 2009). "Alternative splicing and biological heterogeneity in prostate cancer". Nat Rev Urol. 6 (8): 454–460. doi:10.1038/nrurol.2009.125. PMID   19657379. S2CID   30664940.
  19. ↑ Adler, AS; McCleland, ML; Yee, S; Yaylaoglu, M; Hussain, S; Cosino, E; Quinones, G; Modrusan, Z; Seshagiri, S; Torres, E; Chopra, VS; Haley, B; Zhang, Z; Blackwood, EM; Singh, M; Junttila, M; Stephan, JP; Liu, J; Pau, G; Fearon, ER; Jiang, Z; Firestein, R (May 2014). "An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth". Genes Dev. 28 (10): 1068–84. doi:10.1101/gad.237206.113. PMC   4035536 . PMID   24788092.
  20. 1 2 Guo, Rong; Yong Li; Jinying Ning; Dan Sun; Lianjun Lin; Xinmin Liu (2013). "HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells". PLOS ONE. 8 (4): e62729. Bibcode:2013PLoSO...862729G. doi: 10.1371/journal.pone.0062729 . PMC   3639176 . PMID   23658645.
  21. ↑ Acedo, Alberto; David J Sanz; Mercedes Durán; Mar Infante; Lucía Pérez-Cabornero; Cristina Miner; Eladio A Velasco (2012). "Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes". Breast Cancer Research. 14 (3): R87. doi: 10.1186/bcr3202 . PMC   3446350 . PMID   22632462.
  22. ↑ Di Giacomo, D.; Gaildrat, P; Abuli, A; Abdat, J; Frébourg, T; Tosi, M; Martins, A (2013). "Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements". Hum. Mutat. 34 (11): 1547–57. doi: 10.1002/humu.22428 . PMID   23983145. S2CID   22874730.
  23. ↑ Daniotti, Jose L.; Aldo A. Vilcaes; Vanina Torres Demichelis; Fernando M. Ruggiero; Macarena Rodriguez-Walker (2013). "Glycosylation of Glycolipids in Cancer: Basis for Development of Novel Therapeutic Approaches". Front Oncol. 3: 306. doi: 10.3389/fonc.2013.00306 . PMC   3867695 . PMID   24392350.
  24. ↑ Aurisicchio, L; Fridman, A; Bagchi, A; Scarselli, E; La Monica, N; Ciliberto, G (Jan 2014). "A novel minigene scaffold for therapeutic cancer vaccines". Oncoimmunology. 3 (1): e27529. doi:10.4161/onci.27529. PMC   4002591 . PMID   24790791.

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