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The steps contributing to the production of primary transcripts involve a series of molecular interactions that initiate transcription of DNA within a cell's nucleus. Based on the needs of a given cell, certain DNA sequences are transcribed to produce a variety of RNA products to be translated into functional proteins for cellular use. To initiate the transcription process in a cell's nucleus, DNA double helices are unwound and [[hydrogen bond]]s connecting compatible nucleic acids of DNA are broken to produce two unconnected single DNA strands.<ref name="StrachanRead2004">{{cite book| vauthors = Strachan T, Read AP |title=Human Molecular Genetics 3|url=https://books.google.com/books?id=g4hC63UrPbUC|date=January 2004|publisher=Garland Science|isbn=978-0-8153-4184-0|pages=16–17}}</ref> One strand of the DNA template is used for transcription of the single-stranded primary transcript mRNA. This DNA strand is bound by an [[RNA polymerase]] at the [[promoter (genetics)|promoter]] region of the DNA.<ref name="Alberts3rd">{{cite book| vauthors = Alberts B |title=Molecular Biology of the Cell |chapter=RNA Synthesis and RNA Processing | edition = 3rd |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK28319/| via = NCBI |date=1994 |publisher=New York: Garland Science}}</ref>
[[File:Transcription.jpg|thumb|Transcription of DNA by RNA polymerase to produce primary transcript]]
In eukaryotes, three kinds of RNA—[[rRNA]], [[tRNA]], and mRNA—are produced based on the activity of three distinct RNA polymerases, whereas, in [[prokaryotes]], only one RNA polymerase exists to create all kinds of RNA molecules.<ref>{{cite web| vauthors = Griffiths AJ |title=An Introduction to Genetic Analysis |url= https://www.ncbi.nlm.nih.gov/books/NBK21853/|archive-url= https://web.archive.org/web/20200108205509/https://www.ncbi.nlm.nih.gov/books/NBK21853/|url-status= dead|archive-date= January 8, 2020|work=NCBI|date=2000 |publisher=New York: W.H. Freeman}}</ref> RNA polymerase II of eukaryotes transcribes the primary transcript, a transcript destined to be processed into mRNA, from the [[antisense]] DNA template in the 5' to 3' direction, and this newly synthesized primary transcript is complementary to the antisense strand of DNA.<ref name="StrachanRead2004" /> RNA polymerase II constructs the primary transcript using a set of four specific [[ribonucleoside]] monophosphate residues ([[adenosine monophosphate]] (AMP), [[cytidine monophosphate]] (CMP), [[guanosine monophosphate]] (GMP), and [[uridine monophosphate]] (UMP)) that are added continuously to the 3' hydroxyl group on the 3' end of the growing mRNA.<ref name="StrachanRead2004" />
Studies of primary transcripts produced by RNA polymerase II reveal that an average primary transcript is 7,000 [[nucleotide]]s in length, with some growing as long as 20,000 nucleotides in length.<ref name="Alberts3rd"/> The inclusion of both [[exon]] and [[intron]] sequences within primary transcripts explains the size difference between larger primary transcripts and smaller, mature mRNA ready for translation into protein.{{cn|date=July 2024}}
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===Transcription stress===
[[DNA damage (naturally occurring)|DNA damages]] arise in each cell, every day, with the number of damages in each cell reaching tens to hundreds of thousands, and such DNA damages can impede primary transcription.<ref name = Milano2024>{{cite journal |vauthors=Milano L, Gautam A, Caldecott KW |title=DNA damage and transcription stress |journal=Mol Cell |volume=84 |issue=1 |pages=70–79 |date=January 2024 |pmid=38103560 |doi=10.1016/j.molcel.2023.11.014 |url=|doi-access=free }} {{CC-notice|cc=by4}}</ref> The process of [[gene expression]] itself is a source of endogenous DNA damages resulting from the susceptibility of single-stranded DNA to damage.<ref name = Milano2024/> Other sources of DNA damage are conflicts of the primary transcription machinery with the [[DNA replication]] machinery, and the activity of certain enzymes such as [[topoisomerase]]s and [[base excision repair]] enzymes. Even though these processes are tightly regulated and are usually accurate, occasionally they can make mistakes and leave behind DNA breaks that drive [[chromosomal rearrangement]]s or [[cell death]].<ref name = Milano2024/>
==RNA processing==
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===Alternative splicing===
{{Main|Alternative splicing}}
Eukaryotic pre-mRNAs have their introns spliced out by [[spliceosome]]s made up of [[snRNP|small nuclear ribonucleoproteins]].<ref>{{cite book | vauthors = Weaver RF | date = 2005 | title = Molecular Biology | pages =
In complex eukaryotic cells, one primary transcript is able to prepare large amounts of mature mRNAs due to alternative splicing. Alternative splicing is regulated so that each mature mRNA may encode a multiplicity of proteins. [[File:Alternativ splicing.png|thumb|Alternative splicing of the primary transcript]] The effect of alternative splicing in gene expression can be seen in complex eukaryotes which have a fixed number of genes in their genome yet produce much larger numbers of different gene products.<ref name="Cooper GM"/> Most eukaryotic pre-mRNA transcripts contain multiple introns and exons. The various possible combinations of 5' and 3' splice sites in a pre-mRNA can lead to different excision and combination of exons while the introns are eliminated from the mature mRNA. Thus, various kinds of mature mRNAs are generated.<ref name="Cooper GM"/> Alternative splicing takes place in a large protein complex called the [[spliceosome]]. Alternative splicing is crucial for tissue-specific and developmental regulation in gene expression.<ref name="Cooper GM"/> Alternative splicing can be affected by various factors, including mutations such as [[chromosomal translocation]].
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== Research ==
5-[[Fluorouracil]] (FUra) exposure in [[methotrexate]]-resistant KB cells led to a two-fold reduction in total [[dihydrofolate reductase]] (DHFR) mRNA levels, while the level of DHFR pre-mRNA with certain introns remained unaffected. The half-life of DHFR mRNA or pre-mRNA did not change significantly, but the transition rate of DHFR RNA from the nucleus to the cytoplasm decreased, suggesting that FUra may influence mRNA processing and/or nuclear DHFR mRNA stability.<ref>{{cite journal | title = 5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells | journal = The Journal of Biological Chemistry | volume = 264 | issue = 35 | pages = 21413–21 | date = December 1989 | doi = 10.1016/S0021-9258(19)30096-1 | pmid = 2592384 | doi-access = free | vauthors = Will CL, Dolnick BJ }}</ref>
In ''[[Drosophila]]'' and ''[[Aedes]]'', hnRNA (pre-mRNA) size was larger in ''Aedes'' due to its larger genome, despite both species producing mature mRNA of similar size and sequence complexity. This indicates that hnRNA size increases with genome size.<ref>{{cite journal | title = hnRNA size and processing as related to different DNA content in two dipterans: Drosophila and Aedes | journal = Cell | volume = 5 | issue = 3 | pages = 281–90 | date = July 1975 | pmid = 807333 | doi = 10.1016/0092-8674(75)90103-8 | s2cid = 39038640 | vauthors = Lengyel J, Penman S }}</ref>
In [[HeLa cell]]s, spliceosome groups on pre-mRNA were found to form within [[nuclear speckles]], with this formation being temperature-dependent and influenced by specific RNA sequences. Pre-mRNA targeting and splicing factor loading in speckles were critical for spliceosome group formation, resulting in a speckled pattern.<ref>{{cite journal | title = Prespliceosomal assembly on microinjected precursor mRNA takes place in nuclear speckles | journal = Molecular Biology of the Cell | volume = 12 | issue = 2 | pages = 393–406 | date = February 2001 | pmid = 11179423 | doi = 10.1091/mbc.12.2.393 | citeseerx = 10.1.1.324.8865 | pmc = 30951 | vauthors = Melčák I, Melčáková Š, Kopsky V, Večeřová J, Raška I }}</ref>
Recruiting pre-mRNA to nuclear speckles significantly increased splicing efficiency and protein levels, indicating that proximity to speckles enhances splicing efficiency.<ref>{{cite journal | title = Genome organization around nuclear speckles drives mRNA splicing efficiency. | journal = Nature | volume = 629 | issue = 8014 | pages = 1165–1173 | date = May 2024 | pmid = 38720076 | pmc = 11164319 | doi = 10.1038/s41586-024-07429-6 | bibcode = 2024Natur.629.1165B | vauthors = Bhat P, Chow A, Emert B, Ettlin O, Quinodoz SA, Strehle M, Takei Y, Burr A, Goronzy IN, Chen AW, Huang W, Ferrer JL, Soehalim E, Goh S, Chari T, Sullivan DK, Blanco MR, Guttman M }}</ref>
==Related diseases==
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