Primary transcript: Difference between revisions

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{{Short description|RNA produced by transcription}}

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[[File:Pre-mRNA.svg|thumb|Pre-mRNA is the first form of RNA created through transcription in protein synthesis. The pre-mRNA lacks structures that the messenger RNA (mRNA) requires. First all introns have to be removed from the transcribed RNA through a process known as splicing. Before the RNA is ready for export, a Poly(A)tail is added to the 3' end of the RNA and a 5' cap is added to the 5' end.]]

[[File:Transcription label en.jpg|thumb|Micrograph of gene transcription of ribosomal RNA illustrating the growing primary transcripts]]

A '''primary transcript''' is the single-stranded ribonucleic acid ([[RNA]]) product synthesized by [[Transcription (genetics)|transcription]] of [[DNA]], and processed to yield various mature RNA products such as [[mRNA]]s, [[tRNA]]s, and [[rRNA]]s. The primary transcripts designated to be mRNAs are modified in preparation for [[Translation (biology)|translation]]. For example, a '''precursor mRNA''' ('''pre-mRNA''') is a type of primary transcript that becomes a messenger RNA (mRNA) after [[Post-transcriptional modification|processing]].

Pre-mRNA is synthesized from a [[DNA]] template in the [[cell nucleus]] by [[transcription (genetics)|transcription]]. Pre-mRNA comprises the bulk of '''heterogeneous nuclear RNA''' ('''hnRNA'''). Once pre-mRNA has been completely [[Post-transcriptional modification|processed]], it is termed "[[mature messenger RNA]]", or simply "[[messenger RNA]]". The term hnRNA is often used as a synonym for pre-mRNA, although, in the strict sense, hnRNA may include nuclear RNA transcripts that do not end up as cytoplasmic mRNA.

There are several steps contributing to the production of primary transcripts. All these steps involve a series of interactions to initiate and complete the transcription of [[DNA]] in the [[Cell nucleus|nucleus]] of [[eukaryotes]]. Certain factors play key roles in the activation and inhibition of transcription, where they regulate primary transcript production. Transcription produces primary transcripts that are further modified by several processes. These processes include the [[five-prime cap|5' cap]], [[Polyadenylation|3'-polyadenylation]], and [[alternative splicing]]. In particular, alternative splicing directly contributes to the diversity of mRNA found in cells. The modifications of primary transcripts have been further studied in research seeking greater knowledge of the role and significance of these transcripts. Experimental studies based on molecular changes to primary transcripts and the processes before and after transcription have led to greater understanding of diseases involving primary transcripts.

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{{main|Transcription (genetics)}}

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|author1 vauthors =T. Strachan|author2=Andrew P.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/|work 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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A number of factors contribute to the activation and inhibition of transcription and therefore regulate the production of primary transcripts from a given DNA template.{{cn|date=July 2024}}

'''Activation''' of RNA polymerase activity to produce primary transcripts is often controlled by sequences of DNA called [[enhancers]]. [[Transcription factors]], proteins that bind to DNA elements to either activate or repress transcription, bind to enhancers and recruit enzymes that alter [[nucleosome]] components, causing DNA to be either more or less accessible to RNA polymerase. The unique combinations of either activating or inhibiting transcription factors that bind to enhancer DNA regions determine whether or not the gene that enhancer interacts with is activated for transcription or not.<ref name="Gilbert2013">{{cite book|author=Scott F.vauthors = Gilbert SF |title=Developmental Biology|url=https://books.google.com/books?id=-e_bmgEACAAJ|date=15 July 2013|publisher=Sinauer Associates, Incorporated|isbn=978-1-60535-173-5|pages=38–39, 50}}</ref> Activation of transcription depends on whether or not the transcription elongation complex, itself consisting of a variety of transcription factors, can induce RNA polymerase to dissociate from the [[Mediator (coactivator)|Mediator]] complex that connects an enhancer region to the promoter.<ref name="Gilbert2013" /> [[File:Role of transcription factor in gene expression regulation.svg|thumb|Role of transcription factors and enhancers in gene expression regulation]]

'''Inhibition''' of RNA polymerase activity can also be regulated by DNA sequences called [[silencer (DNA)|silencer]]s. Like enhancers, silencers may be located at locations farther up or downstream from the genes they regulate. These DNA sequences bind to factors that contribute to the destabilization of the initiation complex required to activate RNA polymerase, and therefore inhibit transcription.<ref>{{cite book| vauthors = Brown TA | title=Genomes | chapter=Assembly of the Transcription Initiation Complex | date=2002 | edition = 2nd |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK21115/ | ___location = Oxford |publisher= Wiley-Liss}}</ref>

[[Histone]] modification by transcription factors is another key regulatory factor for transcription by RNA polymerase. In general, factors that lead to histone [[acetylation]] activate transcription while factors that lead to histone [[deacetylation]] inhibit transcription.<ref name="Lodish2008">{{cite book|author vauthors =Harvey Lodish H |title=Molecular Cell Biology|url=https://books.google.com/books?id=K3JbjG1JiUMC|year=2008|publisher=W. H. Freeman|isbn=978-0-7167-7601-7|pages=303–306}}</ref> Acetylation of histones induces repulsion between negative components within nucleosomes, allowing for RNA polymerase access. Deacetylation of histones stabilizes tightly coiled nucleosomes, inhibiting RNA polymerase access. In addition to acetylation patterns of histones, methylation patterns at promoter regions of DNA can regulate RNA polymerase access to a given template. RNA polymerase is often incapable of synthesizing a primary transcript if the targeted gene's promoter region contains specific methylated cytosines— residues that hinder binding of transcription-activating factors and recruit other enzymes to stabilize a tightly bound nucleosome structure, excluding access to RNA polymerase and preventing the production of primary transcripts.<ref name="Gilbert2013" />

===R-loops===

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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, RobertRF F.| date = (2005). ''| title = Molecular Biology'', p.432-448.| pages = 432–448 | publisher = McGraw-Hill,| ___location = New York, NY. {{ISBN| isbn = 0-07-284611-9}}.</ref><ref>{{cite journal | vauthors = Wahl MC, Will CL, Lührmann R | title = The spliceosome: design principles of a dynamic RNP machine | journal = Cell | volume = 136 | issue = 4 | pages = 701–18 | date = February 2009 | pmid = 19239890 | doi = 10.1016/j.cell.2009.02.009 | hdl = 11858/00-001M-0000-000F-9EAB-8 | s2cid = 21330280 | hdl-access = free }}</ref>

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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In prokaryotes, splicing is done by [[autocatalytic]] cleavage or by endolytic cleavage. Autocatalytic cleavages, in which no proteins are involved, are usually reserved for sections that code for rRNA, whereas endolytic cleavage corresponds to tRNA precursors.

==Experiments Research ==

A study by Cindy L. Wills and Bruce J. Dolnick from the Department of Experimental Therapeutics at 5-[[Roswell Park Comprehensive Cancer CenterFluorouracil]] (thenFUra) known as the Roswell Park Memorial Institute)exposure in [[Buffalo, New Yorkmethotrexate]]-resistant andKB fromcells the Cell and Molecular Biology Program at University of Wisconsin in Madison, Wisconsin was madeled to understanda cellular processes involving primary transcripts. Researchers wanted to understand whether 5two-[[Fluorouracil]] (FUra), a drug known forfold usereduction in cancer treatment, inhibits or shuts downtotal [[dihydrofolate reductase]] (DHFR) pre-mRNA processing and/or nuclear mRNA stability in [[methotrexate]]-resistant KB cells. Long-term exposure to FUra had no effectlevels, onwhile the level of DHFR pre-mRNA containingwith certain introns, whichremained are sections of pre-mRNA that are usually cut out of the sequence as a part of processingunaffected. However,The levels of total DHFR mRNA decreased two-fold in cells exposed to 1.0 [[micromolar|μM]] FUra. There was no significant change in the [[half-life]], which refers to the time it takes 50% of the mRNA to decay, of total DHFR mRNA or pre-mRNA observeddid innot cellschange exposedsignificantly, tobut FUra.the And nuclear/cytoplasmic RNA labeling experiments demonstrated that thetransition rate of nuclear DHFR RNA changingfrom the nucleus to cytoplasmicthe DHFR mRNAcytoplasm decreased, in cells treated with FUra. These results provide further evidencesuggesting that FUra may helpinfluence in themRNA processing of mRNA precursors and/or affect the stability of nuclear DHFR mRNA stability.<ref>{{cite journal | vauthors = Will CL, Dolnick BJ | 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>

Judith Lengyel and Sheldon Penman from the department of Biology at the [[Massachusetts Institute of Technology]] (MIT) in Cambridge, Massachusetts wrote an article about one type of primary transcript involved in the genes of two [[dipteran]]s, or insects that have two wings:In ''[[Drosophila]]'' and ''[[Aedes]]''. The article describes how researchers looked at, hnRNA, or basically (pre-mRNA, primary transcripts in the two kinds of insects. The) size ofwas hnRNA transcripts and the fraction of hnRNA that is converted to mRNAlarger in cell lines, or groups of cells derived from a single cell of whatever one is studying, of ''Drosophila melanogaster'' and ''Aedes albopictus'' weredue compared. Both insects are dipterans, but ''Aedes'' hasto aits larger genome than ''Drosophila''. This means that Aedes has more DNA, whichdespite meansboth more genes. The ''Aedes'' line make larger hnRNA than did the ''Drosophila'' line even though the two cell lines grew under similar conditions andspecies producedproducing mature or processed mRNA of the samesimilar size and sequence complexity. TheseThis data suggestindicates that thehnRNA size of hnRNA increases with increasing genome size, which is obviously shown by Aedes.<ref>{{cite journal | vauthors = Lengyel J, Penman S | 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>

Ivo Melcak, Stepanka Melcakova, Vojtech Kopsky, Jaromıra Vecerova and Ivan Raska from the department of Cell Biology at the Institute of Experimental Medicine, at the Academy of Sciences of Czech Republic in Prague studied the influences of [[nuclear speckles]] on pre-mRNA. Nuclear speckles (speckles) are a part of the nuclei of cells and are enriched with [[splicing factor]]s known for involvement in mRNA processing. Nuclear speckles have shown to serve neighboring active genes as storage places of these splicing factors. In this study, researchers showed that, in HeLa cells which derived from cells of a person who had cervical cancer and have proven useful for experiments, the first group of [[spliceosome]]s on pre-mRNA come from these speckles. Researchers used microinjections of spliceosome-accepting and mutant [[adenovirus]] pre-mRNAs with differential splicing factor binding to make different groups and then followed the sites in which they were heavily present. Spliceosome-accepting pre-mRNAs were rapidly targeted into the speckles, but the targeting was found to be temperature-dependent. The [[polypyrimidine tract]] sequences in mRNA promote the construction of spliceosome groups and is required for targeting, but, by itself, was not sufficient. The downstream flanking sequences were particularly important for the targeting of the mutant pre-mRNAs in the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides (complementary sequences of DNA and or RNA to a specific sequence) and, in this case, specific sequences of [[snRNA]]s. snRNAs are known for helping in the processing of pre-mRNA as well. Under these conditions, spliceosome groups formed on endogenous pre-mRNAs. Researchers concluded that the spliceosome groups on microinjected pre-mRNA form inside the speckles. Pre-mRNA targeting and buildup in the speckles is a result of the loading of splicing factors to the pre-mRNA, and the spliceosome groups gave rise to the speckled pattern observed.<ref>{{cite journal | vauthors = Melcák I, Melcáková S, Kopský V, Vecerová J, Raska I | 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 }}</ref>

A study by Prashant Bhat and Mitchell Guttman from the Division of Biology and Biological Engineering at the [[California Institute of Technology]] studied whether gene positioning relative to speckles influences splicing outcome. Because highly transcribed, active genes are preferentially organized in the nucleus closer to nuclear speckles, they tested whether splicing efficiency, or the biochemical rate of splicing, is enhanced by a gene being physically close to a speckle. To establish a causal relationship between gene proximity to speckles and splicing efficiency, Bhat and Guttman used an artificial tethering system (MS2-MCP) to drive association of synthetic pre-mRNAs to specific nuclear locations, including speckles. By driving association of theRecruiting pre-mRNA reporter to nuclear speckles, they found that recruitment to speckles significantly boostedincreased splicing efficiency and subsequently protein levels. This result, indicatesindicating that simply recruiting a pre-mRNAproximity to aspeckles speckle is sufficient to increaseenhances splicing efficiency. <ref>{{cite journal | vauthors = Bhat P, Chow A, Emert B et al | 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==

Research has also led to greater knowledge about certain diseases related to changes within primary transcripts. One study involved [[estrogen receptor]]s and differential splicing. The article entitled, "Alternative splicing of the human estrogen receptor alpha primary transcript: mechanisms of exon skipping" by Paola Ferro, Alessandra Forlani, Marco Muselli and Ulrich Pfeffer from the laboratory of Molecular Oncology at National Cancer Research Institute in Genoa, Italy, explains that 1785 nucleotides of the region in the DNA that codes for the estrogen receptor alpha (ER-alpha) are spread over a region that holds more than 300,000 nucleotides in the primary transcript. Splicing of this pre-mRNA frequently leads to variants or different kinds of the mRNA lacking one or more exons or regions necessary for coding proteins. These variants have been associated with [[breast cancer]] progression.<ref>{{cite journal | vauthors = Ferro P, Forlani A, Muselli M, Pfeffer U | title = Alternative splicing of the human estrogen receptor alpha primary transcript: mechanisms of exon skipping | journal = International Journal of Molecular Medicine | volume = 12 | issue = 3 | pages = 355–63 | date = September 2003 | pmid = 12883652 }}</ref> In the life cycle of [[retrovirus]]es, proviral DNA is incorporated in transcription of the DNA of the cell being infected. Since retroviruses need to change their pre-mRNA into DNA so that this DNA can be integrated within the DNA of the host it is affecting, the formation of that DNA template is a vital step for retrovirus replication. Cell type, the differentiation or changed state of the cell, and the physiological state of the cell, result in a significant change in the availability and activity of certain factors necessary for transcription. These variables create a wide range of viral gene expression. For example, tissue culture cells actively producing infectious virions of avian or murine [[leukemia]] viruses (ASLV or MLV) contain such high levels of viral RNA that 5–10% of the mRNA in a cell can be of viral origin. This shows that the primary transcripts produced by these retroviruses do not always follow the normal path to protein production and convert back into DNA in order to multiply and expand.<ref>{{cite book | veditors = Coffin JM, Hughes SH, Varmus HE, editors.| chapter = Transcription | title = Retroviruses. | ___location = Cold Spring Harbor (NY): | publisher = Cold Spring Harbor Laboratory Press; | date = 1997. Available| from:chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK19441/ }}</ref>

== See also ==