Introns and Their Therapeutic Applicationsin Biomedical Researches

Document Type : Review Paper


1 Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

2 Stem Cell and Regenerative Medicine Research Group, Iranian Academic Center for Education, Culture and Research (ACECR), Khorasan Razavi Branch, Mashhad, Iran

3 Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

4 Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

5 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

6 National Institute for Genetic Engineering and Biotechnology, Tehran, Iran


ntext: Although for a long time, it was thought that intervening sequences (introns) were junk DNA without any function, their critical roles and the underlying molecular mechanisms in genome regulation have only recently come to light. 
Introns not only carry information for splicing, but they also play many supportive roles in gene regulation at different 
levels. They are supposed to function as useful tools in various biological processes, particularly in the diagnosis and 
treatment of diseases. Introns can contribute to numerous biological processes, including gene silencing, gene imprinting, 
transcription, mRNA metabolism, mRNA nuclear export, mRNA localization, mRNA surveillance, RNA editing, NMD, 
translation, protein stability, ribosome biogenesis, cell growth, embryonic development, apoptosis, molecular evolution, 
genome expansion, and proteome diversity through various mechanisms.
Evidence Acquisition: In order to fulfill the objectives of this study, the following databases were searched: Medline, Scopus, Web of Science, EBSCO, Open Access Journals, and Google Scholar. Only articles published in English were included.
Results & Conclusions: The intervening sequences of eukaryotic genes have critical functions in genome regulation, 
as well as in molecular evolution. Here, we summarize recent advances in our understanding of how introns influence 
genome regulation, as well as their effects on molecular evolution. Moreover, therapeutic strategies based on intron sequences are discussed. According to the obtained results, a thorough understanding of intron functional mechanisms could 
lead to new opportunities in disease diagnosis and therapies, as well as in biotechnology applications.


Main Subjects

1. De Souza SJ, Long M, Gilbert W. Introns and gene evolution. 
Genes Cells. 1996;1(6):493-505. doi: 10.1046/j.1365-2443. 
2. Kashima T, Rao N, Manley JL. An intronic element contributes 
to splicing repression in spinal muscular atrophy. Proc Natl 
Acad Sci U S A. 2007;104(9):3426-3431. doi: 10.1073/pnas. 
3. Beard WA, Horton JK, Prasad R, Wilson SH. Eukaryotic base 
excision repair: new approaches shine light on mechanism. 
Annu Rev Biophys. 2019;88:137-162. doi: 10.1146/annurev-bio 
4. Poverennaya I, Roytberg M. Spliceosomal introns: features, 
functions, and evolution. Biochemistry (Moscow). 2020;85 
(7):725-734. doi: 10.1134/s0006297920070019
5. Herbert A, Rich A. RNA processing and the evolution of 
eukaryotes. Nat Genet. 1999;21(3):265-269. doi: 10.1038/6780
6. Bouaynaya N, Schonfeld D. The genomic structure: proof of 
the role of non-coding DNA. Conf Proc IEEE Eng Med Biol 
Soc. 2006;1:4544-4547. doi: 10.1109/iembs.2006.259446
7. Rose AB. Introns as gene regulators: a brick on the accelerator. 
Frontiers in genetics. 2019;9:672. doi: 10.3389/fgene.2018. 
8. Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster 
RL. Heterologous introns can enhance expression of transgenes 
in mice. Proc Natl Acad Sci U S A. 1991;88(2):478-482. doi: 
9. Cottrell E, Maharaj A, Williams J, Chatterjee S, Cirillo G, 
Miraglia del Giudice E, et al. Growth Hormone Receptor 
(GHR) 6Ω Pseudoexon Activation: a Novel Cause of Severe 
Growth Hormone Insensitivity. J Clin Endocrinol Met. 2021. 
doi: 10.1210/clinem/dgab550
10. Nesic D, Cheng J, Maquat LE. Sequences within the last intron 
function in RNA 3’-end formation in cultured cells. Mol Cell 
Biol. 1993;13(6):3359-3369. doi: 10.1128/mcb.13.6.3359
11. Antoniou M, Geraghty F, Hurst J, Grosveld F. Efficient 3’-
end formation of human beta-globin mRNA in vivo requires 
sequences within the last intron but occurs independently of 
the splicing reaction. Nucleic Acids Res. 1998;26(3):721-729. 
doi: 10.1093/nar/26.3.721
12. Donath M, Mendel R, Cerff R, Martin W. Intron-dependent 
transient expression of the maize GapA1 gene. Plant Mol Biol. 
1995;28(4):667-676. doi: 10.1007/bf00021192
13. Jia J, Long Y, Zhang H, Li Z, Liu Z, Zhao Y, et al. Posttranscriptional splicing of nascent RNA contributes to 
widespread intron retention in plants. Nature Plants. 
2020;6(7):780-788. doi: 10.1038/s41477-020-0688-1
14. Nott A, Meislin SH, Moore MJ. A quantitative analysis of intron 
effects on mammalian gene expression. RNA. 2003;9(5):607-
617. doi:10.1261/rna.5250403
15. Parenteau J, Maignon L, Berthoumieux M, Catala M, Gagnon 
V, Abou Elela S. Introns are mediators of cell response to 
starvation. Nature. 2019;565(7741):612-617. doi: 10.1038/
16. Liu H, Lyu HM, Zhu K, Van de Peer Y, Cheng ZM. The 
emergence and evolution of intron-poor and intronless 
genes in intron-rich plant gene families. The Plant Journal. 
2021;105(4):1072-1082. doi: 10.1111/tpj.15088
17. Rohrer J, Conley ME. Transcriptional regulatory elements 
within the first intron of Bruton’s tyrosine kinase. Blood. 
1998;91(1):214-221. doi: 10.1182/blood.v91.1.214
18. Furger A, O’Sullivan JM, Binnie A, Lee BA, Proudfoot NJ. 
Promoter proximal splice sites enhance transcription. Genes 
Dev. 2002;16(21):2792-2799. doi: 10.1101/gad.983602
19. Kim DS, Kim TH, Huh JW, Kim IC, Kim SW, Park HS, et al. 
Line Fusion Genes: a database of LINE expression in human 
genes. BMC Genomics. 2006;7:139. doi: 10.1186/1471-2164-
20. Eddy J, Maizels N. Conserved elements with potential to form 
polymorphic G-quadruplex structures in the first intron of 
human genes. Nucleic Acids Res. 2008;36(4):1321-1333. doi: 
21. Tanaka Y, Asano T, Kanemitsu Y, Goto T, Yoshida Y, Yasuba 
K, et al. Positional differences of intronic transposons in 
pAMT affect the pungency level in chili pepper through altered 
splicing efficiency. The Plant Journal. 2019;100(4):693-705. 
doi: 10.1111/tpj.14462
22. Keilwagen J, Hartung F, Grau J. GeMoMa: homology-based 
gene prediction utilizing intron position conservation and 
RNA-seq data. Gene Prediction: Springer; 2019. p. 161-77. 
doi: 10.1007/978-1-4939-9173-0_9
23. Le Hir H, Nott A, Moore MJ. How introns influence and 
enhance eukaryotic gene expression. Trends Biochem Sci. 
2003;28(4):215-220. doi: 10.1016/s0968-0004(03)00052-5
24. Desterro J, Bak-Gordon P, Carmo-Fonseca M. Targeting 
mRNA processing as an anticancer strategy. Nature Reviews 
Drug Discovery. 2020;19(2):112-129. doi: 10.1038/s41573-019- 
25. Jiang W, Geng Y, Liu Y, Chen S, Cao S, Li W, et al. Genomewide identification and characterization of SRO gene family 
in wheat: Molecular evolution and expression profiles during 
different stresses. Plant Physiology and Biochemistry. 
2020;154:590-611. doi: 10.1016/j.plaphy.2020.07.00626. Valadkhan S. snRNAs as the catalysts of pre-mRNA splicing. 
Curr Opin Chem Biol. 2005;9(6):603-608. doi: 10.1016/j.
27. Joynt AT, Evans TA, Pellicore MJ, Davis-Marcisak EF, 
Aksit MA, Eastman AC, et al. Evaluation of both exonic 
and intronic variants for effects on RNA splicing allows for 
accurate assessment of the effectiveness of precision therapies. 
PLoS Gen. 2020;16(10):e1009100. doi: 10.1371/journal.
28. Berget SM, Moore C, Sharp PA. Spliced segments at the 5’ 
terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S 
A. 1977;74(8):3171-3175. doi: 10.1073/pnas.74.8.3171
29. Hua Y, Vickers TA, Okunola HL, Bennett CF, Krainer AR. 
Antisense masking of an hnRNP A1/A2 intronic splicing 
silencer corrects SMN2 splicing in transgenic mice. Am J Hum 
Genet. 2008;82(4):834-848. doi: 10.1016/j.ajhg.2008.01.014
30. Borišek J, Casalino L, Saltalamacchia A, Mays SG, Malcovati 
L, Magistrato A. Atomic-Level Mechanism of Pre-mRNA 
Splicing in Health and Disease. Acc Chem Res. 2020;54(1):144-
154. doi: 10.1021/acs.accounts.0c00578
31. Majewski J, Ott J. Distribution and characterization of 
regulatory elements in the human genome. Genome Res. 
2002;12(12):1827-1836. doi: 10.1101/gr.606402
32. Faustino NA, Cooper TA. Pre-mRNA splicing and human 
disease. Genes Dev. 2003;17(4):419-437. doi: 10.1101/
33. Nilsen TW. The spliceosome: the most complex macromolecular 
machine in the cell? Bioessays. 2003;25(12):1147-1149. doi: 
34. Tang SJ, Shen H, An O, Hong H, Li J, Song Y, et al. Cisand trans-regulations of pre-mRNA splicing by RNA editing 
enzymes influence cancer development. Nature Communicat. 
2020;11(1):1-17. doi: 10.1038/s41467-020-14621-5
35. Tarn WY, Steitz JA. Pre-mRNA splicing: the discovery of a 
new spliceosome doubles the challenge. Trends Biochem Sci. 
1997;22(4):132-137. doi: 10.1016/s0968-0004(97)01018-9
36. Erkelenz S, Poschmann G, Ptok J, Müller L, Schaal H. Profiling 
of cis-and trans-acting factors supporting noncanonical splice 
site activation. RNA Biology. 2021;18(1):118-130. doi: 
37. Tang SJ, Shen H, An O, Hong H, Li J, Song Y, et al. Cisand trans-regulations of pre-mRNA splicing by RNA editing 
enzymes influence cancer development. Nature communicat. 
2020;11(1):799. doi: 10.1038/s41467-020-14621-5
38. Moles-Fernández A, Domènech-Vivó J, Tenés A, Balmaña 
J, Diez O, Gutiérrez-Enríquez S. Role of splicing regulatory 
elements and in silico tools usage in the identification of 
deep intronic splicing variants in hereditary breast/ovarian 
cancer genes. Cancers. 2021;13(13):3341. doi: 10.3390/
39. Finke M, Brecht D, Stifel J, Gense K, Gamerdinger M, Hartig 
JS. Efficient splicing-based RNA regulators for tetracyclineinducible gene expression in human cell culture and C. elegans. 
Nucleic Acids Res. 2021. doi:10.1093/nar/gkab233
40. Monteys AM, Hundley AA, Ranum PT, Tecedor L, Muehlmatt 
A, Lim E, et al. Regulated control of gene therapies by druginduced splicing. Nature. 2021;596(7871):291-295. doi: 
41. Haddad-Mashadrizeh A, Zomorodipour A, Izadpanah M, 
Sam MR, Ataei F, Sabouni F, et al. A systematic study of the 
function of the human beta-globin introns on the expression of 
the human coagulation factor IX in cultured Chinese hamster 
ovary cells. J Gene Med. 2009;11(10):941-950. doi: 10.1002/
42. Hahn S. Structure and mechanism of the RNA polymerase II 
transcription machinery. Nat Struct Mol Biol. 2004;11(5):394-
403. doi: 10.1038/nsmb763
43. Manley JL. Nuclear coupling: RNA processing reaches back 
to transcription. Nat Struct Biol. 2002;9(11):790-791. doi: 10. 
44. Kwek KY, Murphy S, Furger A, Thomas B, O’Gorman W, 
Kimura H, et al. U1 snRNA associates with TFIIH and regulates 
transcriptional initiation. Nat Struct Biol. 2002;9(11):800-805. 
doi: 10.1038/nsb862
45. O’Gorman W, Thomas B, Kwek KY, Furger A, Akoulitchev A. 
Analysis of U1 small nuclear RNA interaction with cyclin H. 
J Biol Chem. 2005;280(44):36920-36925. doi: 10.1074/jbc.m 
46. Tellier M, Maudlin I, Murphy S. Transcription and splicing: 
A two-way street. Wiley Interdisciplinary Reviews: RNA. 
2020;11(5):e1593. doi: 10.1002/wrna.1593
47. Biswas J, Li W, Singer RH, Coleman RA. Imaging 
Organization of RNA Processing within the Nucleus. Cold 
Spring Harbor Perspectives in Biology. 2021:a039453. doi: 
48. Strasser K, Hurt E. Splicing factor Sub2p is required for 
nuclear mRNA export through its interaction with Yra1p. 
Nature. 2001;413(6856):648-652. doi: 10.1038/35098113
49. Reed R, Hurt E. A conserved mRNA export machinery coupled 
to pre-mRNA splicing. Cell. 2002;108(4):523-531. doi: 
50. Stewart M. Polyadenylation and nuclear export of mRNAs. 
J BiologChem. 2019;294(9):2977-2987. doi: 10.1074/jbc.
51. Schell T, Kulozik AE, Hentze MW. Integration of splicing, 
transport and translation to achieve mRNA quality control 
by the nonsense-mediated decay pathway. Genome Biol. 
2002;3(3):Reviews1006. doi: 10.1186/gb-2002-3-3-reviews 
52. Tange TO, Nott A, Moore MJ. The ever-increasing complexities of the exon junction complex. Curr Opin Cell Biol. 
2004;16(3):279-284. doi: 10.1016/
53. Le Hir H, Seraphin B. EJCs at the heart of translational control. 
Cell. 2008;133(2):213-216. doi: 10.1016/j.cell.2008.04.002
54. Kwon OS, Mishra R, Safieddine A, Coleno E, Alasseur Q, 
Faucourt M, et al. Exon junction complex dependent mRNA 
localization is linked to centrosome organization during 
ciliogenesis. Nature communicat. 2021;12(1):1-16. doi: 10. 
55. Woodward LA, Mabin JW, Gangras P, Singh G. The exon 
junction complex: a lifelong guardian of mRNA fate. Wiley 
Interdisciplinary Reviews: RNA. 2017;8(3):e1411. doi: 10.1002/
56. Joseph B, Lai EC. The exon junction complex and intron 
removal prevent re-splicing of mRNA. PLoS Gen. 2021;17 
(5):e1009563. doi: 10.1371/journal.pgen.1009563
57. Mabin JW, Woodward LA, Patton RD, Yi Z, Jia M, Wysocki VH, 
et al. The exon junction complex undergoes a compositional 
switch that alters mRNP structure and nonsense-mediated 
mRNA decay activity. Cell reports. 2018;25(9):2431-2446. e7. 
doi: 10.1016/j.celrep.2018.11.046
58. Herold A, Suyama M, Rodrigues JP, Braun IC, Kutay U, Carmo-Fonseca M, et al. TAP (NXF1) belongs to a multigene 
family of putative RNA export factors with a conserved 
modular architecture. Mol Cell Biol. 2000;20(23):8996-9008. 
doi: 10.1128/mcb.20.23.8996-9008.2000
59. Clouse KN, Luo MJ, Zhou Z, Reed R. A Ran-independent 
pathway for export of spliced mRNA. Nat Cell Biol. 
2001;3(1):97-99. doi: 10.1038/35050625
60. Cheng C, Sharp PA. Regulation of CD44 alternative splicing 
by SRm160 and its potential role in tumor cell invasion. Mol 
Cell Biol. 2006;26(1):362-370. doi: 10.1128/mcb.26.1.362-
61. Hachet O, Ephrussi A. Drosophila Y14 shuttles to the posterior 
of the oocyte and is required for oskar mRNA transport. 
Curr Biol. 2001;11(21):1666-1674. doi: 10.1016/s0960-
62. Le Hir H, Gatfield D, Izaurralde E, Moore MJ. The exonexon junction complex provides a binding platform for factors 
involved in mRNA export and nonsense-mediated mRNA 
decay. EMBO J. 2001;20(17):4987-4997. doi: 10.1093/
63. Shi H, Xu RM. Crystal structure of the Drosophila Mago 
nashi-Y14 complex. Genes Dev. 2003;17(8):971-976. doi: 
64. Lykke-Andersen J, Shu MD, Steitz JA. Communication 
of the position of exon-exon junctions to the mRNA 
surveillance machinery by the protein RNPS1. Science. 
2001;293(5536):1836-1839. doi: 10.1126/science.1062786
65. Maquat LE. Nonsense-mediated mRNA decay: splicing, 
translation and mRNP dynamics. Nat Rev Mol Cell Biol. 
2004;5(2):89-99. doi: 10.1038/nrm1310
66. Makarova JA, Kramerov DA. Noncoding RNA of U87 host 
gene is associated with ribosomes and is relatively resistant to 
nonsense-mediated decay. Gene. 2005;363:51-60. doi: 10.10 
67. Brogna S, Wen J. Nonsense-mediated mRNA decay (NMD) 
mechanisms. Nat Struct Mol Biol. 2009;16(2):107-113. doi: 
68. Ying SY, Lin SL. Intron-derived microRNAs--fine tuning of 
gene functions. Gene. 2004;342(1):25-28. doi: 10.1016/j.
69. Lin SL, Miller JD, Ying SY. Intronic microRNA (miRNA). 
J Biomed Biotechnol. 2006;2006(4):26818. doi: 10.1155/
70. Behl T, Kumar C, Makkar R, Gupta A, Sachdeva M. 
Intercalating the role of microRNAs in cancer: as enemy or 
protector. Asian Pacific journal of cancer prevention: APJCP. 
2020;21(3):593. doi: 10.31557/apjcp.2020.21.3.593
71. Esmailzadeh S, Mansoori B, Mohammadi A, Baradaran B. 
Regulatory roles of micro-RNAs in T cell autoimmunity. 
Immunological investigations. 2017;46(8):864-879. doi: 10. 
72. Hashemzadeh MR. Role of micro RNAs in stem cells, cardiac 
differentiation and cardiovascular diseases. Gene Reports. 
2017;8:11-6. doi: 10.1016/j.genrep.2017.04.012
73. Stark A, Brennecke J, Russell RB, Cohen SM. Identification of 
Drosophila MicroRNA targets. PLoS Biol. 2003;1(3):E60. doi: 
74. Pederson T. RNA interference and mRNA silencing, 2004: 
how far will they reach? Mol Biol Cell. 2004;15(2):407-410. 
doi: 10.1091/mbc.e03-10-0726
75. Tomasello L, Distefano R, Nigita G, Croce CM. The microRNA 
family gets wider: the isomiRs classification and role. Frontiers 
in Cell and Developmental Biology. 2021;9. doi: 10.3389/
76. Ambros V. MicroRNA pathways in flies and worms: growth, 
death, fat, stress, and timing. Cell. 2003;113(6):673-676. doi: 
77. Islam ABMM, Mohammad E, Khan M. Aberration of the 
modulatory functions of intronic microRNA hsa-miR-933 
on its host gene ATF2 results in type II diabetes mellitus and 
neurodegenerative disease development. Human Genomics. 
2020;14(1):1-11. doi: 10.1186/s40246-020-00285-1
78. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, et al. Control of leaf morphogenesis by microRNAs. 
Nature. 2003;425(6955):257-263. doi: 10.1038/nature01958
79. Cao X, Fan Q-L. LncRNA MIR503HG promotes high-glucoseinduced proximal tubular cell apoptosis by targeting miR-503-
5p/bcl-2 pathway. Diabetes, Metabolic Syndrome and Obesity: 
Targets and Therapy. 2020;13:4507. doi: 10.2147/dmso.s277 
80. Qu LH, Henras A, Lu YJ, Zhou H, Zhou WX, Zhu YQ, et 
al. Seven novel methylation guide small nucleolar RNAs are 
processed from a common polycistronic transcript by Rat1p 
and RNase III in yeast. Mol Cell Biol. 1999;19(2):1144-1158. 
doi: 10.1128/mcb.19.2.1144
81. Bachellerie JP, Cavaille J, Huttenhofer A. The expanding snoRNA world. Biochimie. 2002;84(8):775-790. doi: 10.1016/
82. Frazier MN, Pillon MC, Kocaman S, Gordon J, Stanley RE. 
Structural overview of macromolecular machines involved in 
ribosome biogenesis. Current Opinion in Structural Biology. 
2021;67:51-60. doi: 10.1016/
83. Kumar V. Ribosomal biogenesis in eukaryotes. Emerging Concepts in Ribosome Structure, Biogenesis, and Function: 
Elsevier; 2021. p. 129-150. doi: 10.1016/b978-0-12-816364-
84. Bratkovič T, Božič J, Rogelj B. Functional diversity of small 
nucleolar RNAs. Nucleic acids research. 2020;48(4):1627-
1651. doi: 10.1093/nar/gkz1140
85. Huttenhofer A, Kiefmann M, Meier-Ewert S, O’Brien J, 
Lehrach H, Bachellerie JP, et al. RNomics: an experimental 
approach that identifies 201 candidates for novel, small, nonmessenger RNAs in mouse. EMBO J. 2001;20(11):2943-2953. 
doi: 10.1093/emboj/20.11.2943
86. Reinhart BJ, Bartel DP. Small RNAs correspond to centromere 
heterochromatic repeats. Science. 2002;297(5588):1831. doi: 
87. Tritto P, Specchia V, Fanti L, Berloco M, D’Alessandro R, 
Pimpinelli S, et al. Structure, regulation and evolution of the 
crystal-Stellate system of Drosophila. Genetica. 2003;117(2-
3):247-257. doi: 10.1023/a:1022960632306
88. Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette 
MF, et al. Genetic analysis of RNA-mediated transcriptional 
gene silencing. Biochim Biophys Acta. 2004;1677(1-3):129-
141. doi: 10.1016/j.bbaexp.2003.10.015
89. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann 
SA, et al. Functional demarcation of active and silent 
chromatin domains in human HOX loci by noncoding RNAs. 
Cell. 2007;129(7):1311-1323. doi: 10.1016/j.cell.2007.05.022
90. Polavarapu N, Marino-Ramirez L, Landsman D, McDonald 
JF, Jordan IK. Evolutionary rates and patterns for human 
transcription factor binding sites derived from repetitive DNA. BMC Genomics. 2008;9:226. doi: 10.1186/1471-2164-9-226
91. Parris GE. Developmental diseases and the hypothetical Master 
Development Program. Med Hypotheses. 2010;74(3):564-573. 
doi: 10.1016/j.mehy.2009.09.035
92. Qian W, Zhang J. Codon usage bias and nuclear mRNA 
concentration: Correlation vs. causation. Proceedings of the 
National Academy of Sciences. 2021;118(20). doi: 10.1073/
pnas 2104714118
93. Darzacq X, Jady BE, Verheggen C, Kiss AM, Bertrand E, Kiss 
T. Cajal body-specific small nuclear RNAs: a novel class of 
2’-O-methylation and pseudouridylation guide RNAs. EMBO 
J. 2002;21(11):2746-2756. doi: 10.1093/emboj/21.11.2746
94. Nishihara H, Smit AF, Okada N. Functional noncoding 
sequences derived from SINEs in the mammalian genome. 
Genome Res. 2006;16(7):864-874. doi: 10.1101/gr.5255506
95. Huppert JL. Hunting G-quadruplexes. Biochimie. 2008;90(8): 
1140-1148. doi: 10.1016/j.biochi.2008.01.014
96. Vagner S, Vagner C, Mattaj IW. The carboxyl terminus of 
vertebrate poly(A) polymerase interacts with U2AF 65 to 
couple 3’-end processing and splicing. Genes Dev. 2000;14 
(4):403-413. doi: 10.1101/gad.14.4.403
97. Fong YW, Zhou Q. Stimulatory effect of splicing factors on 
transcriptional elongation. Nature. 2001;414(6866):929-933. 
doi: 10.1038/414929a
98. Polak P, Domany E. Alu elements contain many binding sites 
for transcription factors and may play a role in regulation of 
developmental processes. BMC Genomics. 2006;7:133. doi: 
99. Tahiliani J, Leisk J, Aradhya K, Ouyang K, Aradhya S, Nykamp 
K. Utility of RNA Sequencing Analysis in the Context of 
Genetic Testing. Current Genetic Medicine Reports. 2020:1-7. 
doi: 10.1007/s40142-020-00195-7
100. Duquette ML, Handa P, Vincent JA, Taylor AF, Maizels N. 
Intracellular transcription of G-rich DNAs induces formation 
of G-loops, novel structures containing G4 DNA. Genes Dev. 
2004;18(13):1618-1629. doi: 10.1101/gad.1200804
101. Duquette ML, Pham P, Goodman MF, Maizels N. AID binds to 
transcription-induced structures in c-MYC that map to regions 
associated with translocation and hypermutation. Oncogene.
2005;24(38):5791-5798. doi: 10.1038/sj.onc.1208746
102. Duquette ML, Huber MD, Maizels N. G-rich proto-oncogenes 
are targeted for genomic instability in B-cell lymphomas. 
Cancer Res. 2007;67(6):2586-2594. doi: 10.1158/0008-5472.
103. Larson ED, Duquette ML, Cummings WJ, Streiff RJ, Maizels N. 
MutSalpha binds to and promotes synapsis of transcriptionally 
activated immunoglobulin switch regions. Curr Biol. 2005; 
15(5):470-474. doi: 10.1016/j.cub.2004.12.077
104. Burge S, Parkinson GN, Hazel P, Todd AK, Neidle S. 
Quadruplex DNA: sequence, topology and structure. Nucleic 
Acids Res. 2006;34(19):5402-5415. doi: 10.1093/nar/gkl655
105. Maizels N. Dynamic roles for G4 DNA in the biology of 
eukaryotic cells. Nat Struct Mol Biol. 2006;13(12):1055-1059. 
doi: 10.1038/nsmb1171
106. Phan AT, Kuryavyi V, Patel DJ. DNA architecture: from G to 
Z. Curr Opin Struct Biol. 2006;16(3):288-298. doi: 10.1016/j.
107. Yang H, Zhou Y, Liu J. G-quadruplex DNA for construction 
of biosensors. TrAC Trends in Analytical Chemistry. 2020: 
116060. doi: 10.1016/j.trac.2020.116060
108. Huppert JL, Balasubramanian S. Prevalence of quadruplexes 
in the human genome. Nucleic Acids Res. 2005;33(9):2908-
2916. doi: 10.1093/nar/gki609
109. Todd AK, Johnston M, Neidle S. Highly prevalent putative 
quadruplex sequence motifs in human DNA. Nucleic Acids 
Res. 2005;33(9):2901-7. doi: 10.1093/nar/gki553
110. Du Z, Kong P, Gao Y, Li N. Enrichment of G4 DNA motif in 
transcriptional regulatory region of chicken genome. Biochem 
Biophys Res Commun. 2007;354(4):1067-1070. doi: 10.1016/j.
111. Huppert JL, Balasubramanian S. G-quadruplexes in promoters 
throughout the human genome. Nucleic Acids Res. 2007;35(2): 
406-413. doi: 10.1093/nar/gkl1057
112. Zhao Y, Du Z, Li N. Extensive selection for the enrichment of 
G4 DNA motifs in transcriptional regulatory regions of warm 
blooded animals. FEBS Lett. 2007;581(10):1951-1956. doi: 
113. Menon S, Piramanayakam S, Agarwal G. Computational identification of promoter regions in prokaryotes and Eukaryotes. 
EPRA International Journal of Agriculture and Rural Economic 
Research (ARER). 2021;9(7):21-28. doi: 10. 36713/epra7667
114. Haddad-Mashadrizeh A, Hemmat J, Aslamkhan M. Intronic 
regions of the human coagulation factor VIII gene harboring 
transcription factor binding sites with a strong bias towards the 
short-interspersed elements. Heliyon. 2020;6(9):e04727. doi: 
115. Gehring NH, Roignant J-Y. Anything but ordinary–emerging 
splicing mechanisms in eukaryotic gene regulation. Trends in 
Genetics. 2020. doi: 10.1016/j.tig.2020.10.008
116. Petibon C, Malik Ghulam M, Catala M, Abou Elela S. 
Regulation of ribosomal protein genes: An ordered anarchy. 
Wiley Interdisciplinary Reviews RNA. 2021;12(3):e1632. doi: 
117. Lopez AJ. Alternative splicing of pre-mRNA: developmental 
consequences and mechanisms of regulation. Annu Rev Genet. 
1998;32:279-305. doi: 10.1146/annurev.genet.32.1.279
118. Modrek B, Lee C. A genomic view of alternative splicing. Nat 
Genet. 2002;30(1):13-19. doi: 10.1038/ng0102-13
119. Bruno IG, Jin W, Cote GJ. Correction of aberrant FGFR1 
alternative RNA splicing through targeting of intronic 
regulatory elements. Hum Mol Genet. 2004;13(20):2409-2420. 
doi: 10.1093/hmg/ddh272
120. Ladomery MR, Harper SJ, Bates DO. Alternative splicing 
in angiogenesis: the vascular endothelial growth factor 
paradigm. Cancer Lett. 2007;249(2):133-142. doi: 10.1016/j.
121. Lamaa A, Humbert J, Aguirrebengoa M, Cheng X, Nicolas 
E, Côté J, et al. Integrated analysis of H2A. Z isoforms 
function reveals a complex interplay in gene regulation. Elife. 
2020;9:e53375. doi: 10.7554/elife.53375
122. Sun H, Chasin LA. Multiple splicing defects in an intronic 
false exon. Mol Cell Biol. 2000;20(17):6414-6425. doi: 
123. Vela E, Roca X, Isamat M. Identification of novel splice variants 
of the human CD44 gene. Biochem Biophys Res Commun. 
2006;343(1):167-170. doi: 10.1016/j.bbrc.2009.06.049
124. Di Segni G, Gastaldi S, Tocchini-Valentini GP. Cis- and transsplicing of mRNAs mediated by tRNA sequences in eukaryotic 
cells. Proc Natl Acad Sci U S A. 2008;105(19):6864-6869. doi: 
125. Viles KD, Sullenger BA. Proximity-dependent and proximityindependent trans-splicing in mammalian cells. RNA. 2008;(6):1081-1094. doi: 10.1261/rna.384808
126. Hasler J, Strub K. Alu elements as regulators of gene 
expression. Nucleic Acids Res. 2006;34(19):5491-5497. doi: 
127. Bhadra M, Howell P, Dutta S, Heintz C, Mair WB. Alternative splicing in aging and longevity. Human genetics. 2020;139 
(3):357-369. doi: 10.1007/s00439-019-02094-6
128. Sorek R, Ast G, Graur D. Alu-containing exons are alternatively 
spliced. Genome Res. 2002;12(7):1060-1067. doi: 10.1101/gr. 
129. Pérez-Molina R, Arzate-Mejía RG, Ayala-Ortega E, Guerrero 
G, Meier K, Suaste-Olmos F, et al. An intronic Alu element 
attenuates the transcription of a long non-coding RNA in 
human cell lines. Frontiers In Genetics. 2020;11:928. doi: 10. 
130. Lozano G, Francisco-Velilla R, Martinez-Salas E. Deconstructing internal ribosome entry site elements: an update of 
structural motifs and functional divergences. Royal Society 
Open Biology. 2018;8(11):180155. doi: 10.1098/rsob.180155
131. Babich V, Aksenov N, Alexeenko V, Oei SL, Buchlow G, 
Tomilin N. Association of some potential hormone response 
elements in human genes with the Alu family repeats. Gene. 
1999;239(2):341-349. doi: 10.1016/s0378-1119(99)00391-1
132. Li W, Kuzoff R, Wong CK, Tucker A, Lynch M. Characterization 
of newly gained introns in Daphnia populations. Genome 
biology and evolution. 2014;6(9):2218-2234. doi: 10.1093/
133. Makalowski W. Genomic scrap yard: how genomes utilize all 
that junk. Gene. 2000;259(1-2):61-67. doi: 10.1016/s0378-
134. Nekrutenko A, Li WH. Transposable elements are found in a 
large number of human protein-coding genes. Trends Genet. 
2001;17(11):619-621. doi: 10.1016/s0168-9525(01)02445-3
135. Corley M, Flynn RA, Lee B, Blue SM, Chang HY, Yeo GW. 
Footprinting SHAPE-eCLIP Reveals Transcriptome-wide 
Hydrogen Bonds at RNA-Protein Interfaces. Molecular Cell. 
2020;80(5):903-914. e8. doi: 10.1016/j.molcel.2020.11.014
136. Jo B-S, Choi SS. Introns: the functional benefits of introns 
in genomes. Genomics & informatics. 2015;13(4):112. doi: 
137. Baralle FE, Giudice J. Alternative splicing as a regulator of 
development and tissue identity. Nature Rev Molr cell biolog. 
2017;18(7):437-451. doi: 10.1038/nrm.2017.27
138. Chang YF, Imam JS, Wilkinson MF. The nonsensemediated decay RNA surveillance pathway. Annu Rev 
Biochem. 2007;76:51-74. doi: 10.1146/annurev.biochem.76. 
139. Hagiwara M. Alternative splicing: a new drug target of the 
post-genome era. Biochim Biophys Acta. 2005;1754(1-2):324-
331. doi: 10.1016/j.bbapap.2005.09.010
140. Wei C, Xie W, Huang X, Mo X, Liu Z, Wu G, et al. Profiles 
of alternative splicing events in the diagnosis and prognosis 
of Gastric Cancer. J Cancer. 2021;12(10):2982. doi: 10.7150/
141. Eblen ST. Extracellular-regulated kinases: signaling from Ras 
to ERK substrates to control biological outcomes. Adv Cancer 
Res. 2018;138:99-142. doi: 10.1016/bs.acr.2018.02.004
142. Hujová P, Souček P, Grodecká L, Grombiříková H, Ravčuková 
B, Kuklínek P, et al. Deep intronic mutation in SERPING1 
caused hereditary angioedema through pseudoexon activation. 
Journal of clinical immunology. 2020;40(3):435-46. doi: 
143. Venables JP. Aberrant and alternative splicing in cancer. 
Cancer Res. 2004;64(21):7647-5764. doi: 10.1158/0008-5472.
144. Venables JP. Unbalanced alternative splicing and its 
significance in cancer. Bioessays. 2006;28(4):378-386. doi: 
145. Rhine CL, Cygan KJ, Soemedi R, Maguire S, Murray MF, 
Monaghan SF, et al. Hereditary cancer genes are highly susceptible 
to splicing mutations. PLoS Gen. 2018;14(3):e1007231. doi: 
146. Kashkan I, Timofeyenko K, Kollárová E, Růžička K. In Vivo
Reporters for Visualizing Alternative Splicing of Hormonal 
Genes. Plants. 2020;9(7):868. doi: 10.3390/plants9070868
147. Biamonti G, Infantino L, Gaglio D, Amato A. An intricate 
connection between alternative splicing and phenotypic 
plasticity in development and cancer. Cells. 2020;9(1):34. doi: 
148. Sneath RJ, Mangham DC. The normal structure and function 
of CD44 and its role in neoplasia. Mol Pathol. 1998;51(4):191-
200. doi: 10.1136/mp.51.4.191
149. Chalfant CE, Rathman K, Pinkerman RL, Wood RE, Obeid LM, 
Ogretmen B, et al. De novo ceramide regulates the alternative 
splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma 
cells. Dependence on protein phosphatase-1. J Biol Chem. 
2002;277(15):12587-12595. doi: 10.1074/jbc.m112010200
150. Makhafola TJ, Mbele M, Yacqub-Usman K, Hendren A, Haigh 
DB, Blackley Z, et al. Apoptosis in cancer cells is induced by 
alternative splicing of hnRNPA2/B1 through splicing of Bcl-x, 
a mechanism that can be stimulated by an extract of the South 
African Medicinal Plant, Cotyledon orbiculata. Frontiers in 
Oncology. 2020;10. doi: 10.3389/fonc.2020.547392
151. Blake D, Lynch KW. The three as: Alternative splicing, 
alternative polyadenylation and their impact on apoptosis in 
immune function. Immunol Rev. 2021. doi: 10.1111/imr.13018
152. López-Martínez A, Soblechero-Martín P, de-la-Puente-Ovejero 
L, Nogales-Gadea G, Arechavala-Gomeza V. An overview of 
alternative splicing defects implicated in myotonic dystrophy 
type i. Genes. 2020;11(9):1109. doi: 10.3390/genes11091109
153. Hofmann Y, Lorson CL, Stamm S, Androphy EJ, Wirth B. 
Htra2-beta 1 stimulates an exonic splicing enhancer and can 
restore full-length SMN expression to survival motor neuron 2 
(SMN2). Proc Natl Acad Sci U S A. 2000;97(17):9618-9623. 
doi: 10.1073/pnas.160181697
154. Nissim-Rafinia M, Chiba-Falek O, Sharon G, Boss A, Kerem 
B. Cellular and viral splicing factors can modify the splicing 
pattern of CFTR transcripts carrying splicing mutations. Hum 
Mol Genet. 2000;9(12):1771-1778. doi: 10.1093/hmg/9.12. 
155. Helman G, Takanohashi A, Hagemann TL, Perng MD, Walkiewicz 
M, Woidill S, et al. Type II Alexander disease caused by splicing 
errors and aberrant overexpression of an uncharacterized GFAP 
isoform. Human mutation. 2020;41(6):1131-1137. doi: 10.1002/
156. Sazani P, Kole R. Modulation of alternative splicing by antisense 
oligonucleotides. Prog Mol Subcell Biol. 2003;31:217-239. 
doi: 10.1007/978-3-662-09728-1_8
157. Celotto AM, Lee JW, Graveley BR. Exon-specific RNA 
interference: a tool to determine the functional relevance of 
proteins encoded by alternatively spliced mRNAs. Methods 
Mol Biol. 2005;309:273-282. doi: 10.1385/1-59259-935-4:273158. Scharner J, Ma WK, Zhang Q, Lin K-T, Rigo F, Bennett CF, 
et al. Hybridization-mediated off-target effects of spliceswitching antisense oligonucleotides. Nucleic Acids Res. 
2020;48(2):802-816. doi: 10.1093/nar/gkz1132
159. Halloy F, Iyer PS, Ćwiek P, Ghidini A, Barman-Aksözen J, 
Wildner-Verhey van Wijk N, et al. Delivery of oligonucleotides 
to bone marrow to modulate ferrochelatase splicing in a mouse 
model of erythropoietic protoporphyria. Nucleic Acids Res. 
2020;48(9):4658-71. doi: 10.1093/nar/gkaa229
160. Pilch B, Allemand E, Facompre M, Bailly C, Riou JF, Soret J, 
et al. Specific inhibition of serine- and arginine-rich splicing 
factors phosphorylation, spliceosome assembly, and splicing 
by the antitumor drug NB-506. Cancer Res. 2001;61(18):6876-
161. Chen Y, Huang M, Liu X, Huang Y, Liu C, Zhu J, et al. 
Alternative splicing of mRNA in colorectal cancer: new 
strategies for tumor diagnosis and treatment. Cell Death & 
Disease. 2021;12(8):1-16. doi: 10.1038/s41419-021-04031-w
162. Varani L, Spillantini MG, Goedert M, Varani G. Structural 
basis for recognition of the RNA major groove in the tau exon 
10 splicing regulatory element by aminoglycoside antibiotics. 
Nucleic Acids Res. 2000;28(3):710-719. doi: 10.2210/pdb1ei2/
163. Liu X, Jiang Q, Mansfield SG, Puttaraju M, Zhang Y, Zhou 
W, et al. Partial correction of endogenous DeltaF508 CFTR 
in human cystic fibrosis airway epithelia by spliceosomemediated RNA trans-splicing. Nat Biotechnol. 2002;20(1):47-
52. doi: 10.1038/nbt0102-47
164. Lu S, Cullen BR. Analysis of the stimulatory effect of splicing 
on mRNA production and utilization in mammalian cells. 
RNA. 2003;9(5):618-630. doi: 10.1261/rna.5260303
165. Sam MR, Zomorodipour A, Shokrgozar MA, Ataei F, 
Haddad-Mashadrizeh A, Amanzadeh A. Enhancement of the 
human factor IX expression, mediated by an intron derived 
fragment from the rat aldolase B gene in cultured hepatoma 
cells. Biotechnol Lett. 2010;32(10):1385-1392. doi: 10.1007/
166. Appledorn DM, Patial S, McBride A, Godbehere S, Van 
Rooijen N, Parameswaran N, et al. Adenovirus vector-induced 
innate inflammatory mediators, MAPK signaling, as well as 
adaptive immune responses are dependent upon both TLR2 
and TLR9 in vivo. J Immunol. 2008;181(3):2134-2144. doi: 
167. Tang R, Xu Z. Gene therapy: A double-edged sword with great 
powers. Mol Cell Biochem. 2020;474(1):73-81. doi: 10.1007/
168. Jiao Y, Xia ZL, Ze LJ, Jing H, Xin B, Fu S. Research Progress 
of nucleic acid delivery vectors for gene therapy. Biomedical 
microdevices. 2020;22(1):1-10. doi: 10.1007/s10544-020-0469-7
169. Schuppe HC, Meinhardt A. Immune privilege and inflammation 
of the testis. Chem Immunol Allergy. 2005;88:1-14. doi: 10. 
170. Willerth SM, Sakiyama-Elbert SE. Combining stem cells and 
biomaterial scaffolds for constructing tissues and cell delivery. 
2008. doi: 10.3824/stembook.1.1.1
171. Haoudi A, Semmes OJ, Mason JM, Cannon RE. Retrotransposition-Competent Human LINE-1 Induces Apoptosis in 
Cancer Cells With Intact p53. J Biomed Biotechnol. 2004; 
2004(4):185-194. doi: 10.1155/s1110724304403131
172. Yang Y, Walsh CE. Spliceosome-mediated RNA transsplicing. Mol Ther. 2005;12(6):1006-1012. doi: 10.1016/j.ymthe. 
173. Chao H, Walsh CE. RNA repair for haemophilia A. Expert Rev 
Mol Med. 2006;8(1):1-8. doi: 10.1017/S1462399406010337
174. Wood M, Yin H, McClorey G. Modulating the expression 
of disease genes with RNA-based therapy. PLoS Genet. 
2007;3(6):e109. doi: 10.1371/journal.pgen.0030109
175. Wang J, Mansfield SG, Cote CA, Jiang PD, Weng K, Amar MJ, 
et al. Trans-splicing into highly abundant albumin transcripts 
for production of therapeutic proteins in vivo. Mol Ther. 
2009;17(2):343-351. doi: 10.1038/mt.2008.260
176. To TK, Nishizawa Y, Inagaki S, Tarutani Y, Tominaga S, Toyoda 
A, et al. RNA interference-independent reprogramming of DNA 
methylation in Arabidopsis. Nature Plants. 2020;6(12):1455-
1467. doi: 10.1038/s41477-020-00810-z
177. Hong EM, Ingemarsdotter CK, Lever AM. Therapeutic 
applications of trans-splicing. British Medical Bulletin. 
2020;136(1):4-20. doi: 10.1093/bmb/ldaa028
178. Riedmayr LM. SMaRT for therapeutic purposes. Chimeric 
RNA: Springer; 2020. p. 219-232. doi: 10.1007/978-1-4939-
179. Luo M-J, Zhou Z, Magni K, Christoforides C, Rappsilber J, 
Mann M, et al. Pre-mRNA splicing and mRNA export linked 
by direct interactions between UAP56 and Aly. Nature. 
2001;413(6856):644-647. doi: 10.1038/35098106
180. Anderson CM, Kohorn BD. Inactivation of Arabidopsis SIP1 
leads to reduced levels of sugars and drought tolerance. J 
Plant Physiolog. 2001;158(9):1215-1219. doi: 10.1078/s0176-
181. Besse F, Ephrussi A. Translational control of localized 
mRNAs: restricting protein synthesis in space and time. Nat 
Rev Mol Cell Biol. 2008;9(12):971-980. doi: 10.1038/nrm2548
182. Gustafsson C, Reid R, Greene PJ, Santi DV. Identification 
of new RNA modifying enzymes by iterative genome search 
using known modifying enzymes as probes. Nucleic Acids Res. 
1996;24(19):3756-3762. doi: 10.1093/nar/24.19.3756
183. Liu J, Perumal NB, Oldfield CJ, Su EW, Uversky VN, Dunker 
AK. Intrinsic disorder in transcription factors. Biochemistry. 
2006;45(22):6873-6888. doi: 10.1021/bi0602718
184. Cooke C, Hans H, Alwine JC. Utilization of splicing elements 
and polyadenylation signal elements in the coupling of 
polyadenylation and last-intron removal. Mol Cell Biol. 
1999;19(7):4971-4979. doi: 10.1128/mcb.19.7.4971