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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. Pre mRNA is synthesized from a DNA template in the cell nucleus by transcription. Pre mRNA comprises the bulk of heterogeneous nuclear RNA (hnRNA). Once pre mRNA has been completely 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 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 5' cap, 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.

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 bonds connecting compatible nucleic acids of DNA are broken to produce two unconnected single DNA strands. 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 region of the DNA. 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. 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. 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 complex that connects an enhancer region to the promoter. 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. 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.

Transcription, a highly regulated phase in gene expression, produces primary transcripts. However, transcription is only the first step which should be followed by many modifications that yield functional forms of RNAs. Otherwise stated, the newly synthesized primary transcripts are modified in several ways to be converted to their mature, functional forms to produce different proteins and RNAs such as mRNA, tRNA, and rRNA.

The basic primary transcript modification process is similar for tRNA and rRNA in both eukaryotic and prokaryotic cells. On the other hand, primary transcript processing varies in mRNAs of prokaryotic and eukaryotic cells. 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. 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. 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 dipterans, or insects that have two wings: 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 of hnRNA transcripts and the fraction of hnRNA that is converted to mRNA in cell lines, or groups of cells derived from a single cell of whatever one is studying, of Drosophila melanogaster and Aedes albopictus were compared. Both insects are dipterans, but Aedes has a larger genome than Drosophila. This means that Aedes has more DNA, which means more genes. The Aedes line make larger hnRNA than did the Drosophila line even though the two cell lines grew under similar conditions and produced mature or processed mRNA of the same size and sequence complexity. These data suggest that the size of hnRNA increases with increasing genome size, which is obviously shown by Aedes. 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 factors 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 spliceosomes 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 snRNAs. 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.

Research has also led to greater knowledge about certain diseases related to changes within primary transcripts. One study involved estrogen receptors 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. In the life cycle of retroviruses, 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.