In prokaryotic organisms, the primary transcripts of mRNA encoding genes begin to serve as translation templates even before their transcription has been completed. This can occur because the site of transcription is not compartmentalized into the nucleus as it is in eukaryotic organisms. Thus, transcription and translation are coupled in prokaryotic cells. Consequently, prokaryotic mRNAs are subjected to little processing prior to carrying out their intended function in protein synthesis. Indeed, appropriate regulation of some genes (eg, the Trp operon) relies on this coupling of transcription and translation. Prokaryotic rRNA and tRNA molecules are transcribed in units considerably longer than the ultimate molecule. In fact, many of the tRNA transcription units encode more than one tRNA molecule. Thus, in prokaryotes, the processing of these rRNA and tRNA precursor molecules is required for the generation of the mature functional molecules.

Nearly all eukaryotic RNA primary transcripts undergo extensive processing between the time they are synthesized and the time at which they serve their ultimate function, whether it be as mRNA, miRNAs, or as a component of the translation machinery such as rRNA or tRNA. Processing occurs primarily within the nucleus. The processes of transcription, RNA processing, and even RNA transport from the nucleus are highly coordinated. Indeed, a transcriptional coactivator termed SAGA in yeasts and P/CAF in human cells is thought to link transcription activation to RNA processing by recruiting a second complex termed transcription export (TREX) to transcription elongation, splicing, and nuclear export. TREX represents a likely molecular link between transcription elongation complexes, the RNA splicing machinery, and nuclear export (Figure 1). This coupling is thought to dramatically increase both the fidelity and rate of processing and movement of mRNA to the cytoplasm for translation.

Fig1. RNA polymerase II−mediated mRNA gene transcription is cotranscriptionally coupled to RNA processing and transport. Shown (left) is RNA pol II actively transcribing an mRNA encoding gene (elongation top to bottom of figure). RNA processing factors (ie, SR/RRM motif-containing splicing factors as well as polyadenylation and termination factors) interact with the C-terminal domain (CTD, composed of multiple copies of a heptapeptide with consensus sequence –YSPTSPS-) of pol II, while mRNA packaging and transport factors such as TREX complex (pink ovals) are recruited to the nascent mRNA primary transcript, either through direct pol II interactions as shown or, through interactions with SR/splicing factors (brown circles) resident on the nascent mRNA precursor molecules. Note that the CTD is not drawn to scale. The evolutionarily conserved CTD of the Rpb1 subunit of pol II is in reality 5 to 10 times the length of the polymerase due to its many prolines and consequent unstructured nature, and thus a significant docking site for RNA processing and transport proteins among many other critical mRNA metabolizing activities (ie, mRNA polyadenylation and transcription termination factors). In both cases, nascent mRNA chains are thought to be more rapidly and accurately processed due to the immediate recruitment of these many factors to the growing mRNA (precursor) chain. Following appropriate mRNA processing, the mature mRNA is delivered to the nuclear pores dotting the nuclear membrane, where, upon transport through the pores, the mRNAs can be engaged by ribosomes and translated into protein. https://allebookstores.com

The Coding Portions (Exons) of Most Eukaryotic mRNA Encoding Genes Are Interrupted by Introns

 The RNA sequences that appear in mature RNAs are termed exons. In mRNA encoding genes these exons are often interrupted by long sequences of DNA that neither appear in mature mRNA, nor contribute to the genetic information ultimately translated into the amino acid sequence of a protein molecule. In fact, these sequences often interrupt the coding region of protein-encoding genes. These intervening sequences, or introns, exist within most but not all mRNA encoding genes of higher eukaryotes. Human mRNA-encoding gene exons average ~150 nt, while introns are much more heterogeneous, ranging from 10 to 30,000 nucleotides in length. The largest characterized intron in humans is 1,100,000 nt and resides within the 1,586,329 bp KCNIP4 gene (Genebank Record: NC000004.12), which encodes the potassium voltage-gated channel interacting protein 4. Intron RNA sequences are cleaved out of pre-mRNA transcripts, and the exons of the transcript are appropriately spliced together in the nucleus before the resulting mRNA molecule is transported to the cytoplasm for translation (Figures 2 and 3).

Fig2. The processing of the primary transcript to mRNA. In this hypothetical transcript, the 5′ (left) end of the intron is cut (→) and a structure resembling a lariat forms between the G at the 5′ end of the intron and an A near the 3′ end, in the consensus sequence UACUAAC. This sequence is called the branch site, and it is the 3′ most A that forms the 5′–2′ bond with the G. The 3′ (right) end of the intron is then cut (⇓). This releases the lariat, which is digested, and exon 1 is joined to exon 2 at G residues.

Fig3. Consensus sequences at splice junctions. The 5′ (donor; left) and 3′ (acceptor; right) consensus sequences are shown. Also shown is the yeast consensus sequence (UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPyPuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The site of branch formation (ie, Figure 2) is located 20 to 40 nucleotides upstream from the 3′–splice site.

Introns Are Removed & Exons Are Spliced Together

 Several different splicing reaction mechanisms for intron removal have been described. The one most frequently used in eukaryotic cells is described later. Although the sequences of nucleotides in the introns of the various eukaryotic transcripts— and even those within a single transcript—are quite heterogeneous, there are reasonably conserved sequences at each of the two exons–intron (splice) junctions and at the branch site, which is located 20 to 40 nucleotides upstream from the 3′–splice site (see consensus sequences in Figure 3). A special multi component complex, the spliceosome, is involved in converting the primary transcript into mRNA. Spliceosomes consist of the primary transcript, five snRNAs (U1, U2, U4, U5, and U6), and more than 60 proteins, many of which contain conserved RRM (RNA recognition) and SR (serine–arginine) protein motifs. Collectively, the five snRNAs and RRM/SR-containing proteins form a small nuclear ribonucleoprotein termed an snRNP complex. It is likely that this penta-snRNP spliceosome forms prior to interaction with mRNA precursors. snRNPs are thought to position the exon and intron RNA segments for the necessary splicing reactions. The splicing reaction starts with a cut at the junction of the 5′ exon (donor on left) and intron (see Figure2). This is accomplished by a nucleophilic attack by an adenylyl residue in the branch point sequence located just upstream from the 3′end of this intron. The free 5′terminus then forms a loop or lariat structure that is linked by an unusual 5′–2′ phosphodiester bond to the reactive A in the PyNPyPyPuAPy branch site sequence (see Figure 3). This adenylyl residue is typically located 20 to 30 nucleotides upstream from the 3′ end of the intron being removed. The branch site identifies the 3′–splice site. A second cut is made at the junction of the intron with the 3′ exon (donor on right). In this second transesterification reaction, the 3′ hydroxyl of the upstream exon attacks the 5′ phosphate at the downstream exon–intron boundary and the lariat structure containing the intron is released and hydrolyzed. The 5′ and 3′ exons are ligated to form a continuous sequence.

The snRNAs and associated proteins are required for formation of the various structures and intermediates. U1 within the snRNP complex binds first by base pairing to the 5′ exon–intron boundary. U2 within the snRNP complex then binds by base pairing to the branch site, and this exposes the nucleophilic A residue. U4/U5/U6 within the snRNP complex mediates an ATP-dependent protein-mediated unwinding that results in disruption of the base-paired U4–U6 complex with the release of U4. U6 is then able to interact first with U2, then with U1. These interactions serve to approximate the 5′–splice site, the branch point with its reactive A, and the 3′–splice site. Alignment is enhanced by U5. This process also results in the formation of the loop or lariat structure. The two ends are cleaved by the U2–U6 within the snRNP complex. It is important to note that RNA (in concert with two specifically bound Mg2+ions) serves as the catalytic agent. This sequence of events is then repeated in genes containing multiple introns. In such cases, a definite pattern is followed for each gene, though the introns are not necessarily removed in sequence—1, then 2, then 3, etc. Recent high-resolution cryoelectron microscopy (cryo-EM) studies have elucidated the structures of various states of the spliceosome in action.

Alternative Splicing Provides for Production of Different mRNAs From a Single mRNA Primary Transcript, Thereby Increasing the Genetic Potential of an Organism

 The processing of mRNA molecules is a site for regulation of gene expression. Alternative patterns of mRNA splicing result from tissue-specific adaptive and developmental control mechanisms. Interestingly, recent studies suggest that alternative splicing is controlled, at least in part, through chroma tin epigenetic marks (ie, Table 1). This form of coupling of transcription and mRNA processing may either be kinetic and/or mediated through interactions between specific his tone PTMs and alternative splicing factors that can load onto nascent mRNA gene transcripts during the process of transcription.

Table1. Classes of Eukaryotic RNA

As mentioned earlier, the sequence of exon–intron splicing events generally follows a hierarchical order for a given gene. The fact that very complex RNA structures are formed during splicing—and that a number of snRNAs and proteins are involved—affords numerous possibilities for a change of this order and for the generation of different mRNAs. Similarly, the use of alternative termination-cleavage polyadenylation sites also results in mRNA variability. Some schematic examples of these processes, all of which have been well documented in a large array of organisms, including humans, are shown in Figure 4.

Fig4. Mechanisms of alternative processing of mRNA precursors. This form of mRNA processing involves the selective inclusion or exclusion of exons, the use of alternative 5′–donor or 3′–acceptor sites, and the use of different polyadenylation sites, and dramatically increases the differential protein coding potential of the genome.

Not surprisingly, defects in mRNA splicing can cause disease. One of the first examples of the critical importance of accurate splicing was the discovery that one form of β-thalassemia, a disease in which the β-globin gene of hemoglobin is severely under-expressed, results from a nucleotide change at an exon–intron junction. This mutation precludes removal of the intron, altering the translational reading frame of β-globin mRNA, thereby blocking β-chain protein production, and hence hemoglobin. Note that all of these mechanisms rep resent control of gene expression post- or after transcription. Such mutations function in thecis-configuration—that is only the mutated allele, carrying the mutated β-globin gene will be defective in β-globin protein production. Not surprisingly, mutations in a/the gene that encodes the proteins that catalyze splicing and/or polyadenylation can also cause defects in splicing and RNA metabolism, and hence disease. Because these altered proteins will affect the formation/function of many, or all mRNAs, such variants are said to act in trans.

Alternative Promoter Utilization Also Provides a Form of Regulation

Tissue-specific regulation of gene expression can be provided by alternative splicing, as noted earlier, by regulatory DNA control elements in the promoter, or by the use of alternative promoters. The glucokinase (GK) gene consists of 10 exons interrupted by 9 introns. The sequence of exons 2 to 10 is identical in liver and pancreatic β cells, the primary tissues in which GK protein is expressed. Expression of the GK gene is regulated very differently—by two different promoters—in these two tis sues. The liver promoter and exon 1L are located near exons 2 to 10; exon 1L is ligated directly to exon 2. By contrast, the pancreatic β-cell promoter is located about 30-kbp upstream. In this case, the 3′ boundary of exon 1β is ligated to the 5′ boundary of exon 2. The liver promoter and exon 1L are excluded and removed during the splicing reaction (Figure 5). The existence of multiple distinct promoters allows for cell- and tissue-specific expression patterns of a particular gene (mRNA). In the case of GK, insulin and cAMP (see Chapter 42) control GKtranscription in liver, while glucose controls GK expression in β cells. Moreover, as noted earlier, such variation in spliced mRNAs can also change the protein coding protein potential of these mRNAs.

Fig5. Alternative promoter use in the liver and pancreatic β-cell glucokinase (GK) genes. Differential regulation of the glucokinase gene is accomplished by the use of tissue-specific promoters. The β-cell GK gene promoter and exon 1β are located about 30-kbp upstream from the liver promoter and exon 1L. Each promoter has a unique structure and is regulated differently. Exons 2 to 10 are identical in the two genes, and the GK proteins encoded by the liver and β-cell mRNAs have identical kinetic properties.

Both Ribosomal RNAs & Most Transfer RNAs Are Processed From Larger Precursors

In mammalian cells three rRNA molecules (28S, 18S, 5.8S) are transcribed as part of a single large 45S precursor molecule. The precursor is subsequently processed in the nucleolus to provide these three RNA components for the ribosome sub units found in the cytoplasm. The rRNA genes are located in the nucleoli of mammalian cells. Hundreds of copies of these genes are present in every cell. This large number of genes is needed to synthesize sufficient copies of each type of rRNA to form the 10⁷ ribosomes required for every round of cell duplication. Whereas a single mRNA molecule may be copied into 10⁵ protein molecules, providing a large amplification, the rRNAs are end products. This lack of amplification requires both a large number of genes and a high transcription rate, typically synchronized with cell growth rate. Similarly, tRNAs are often synthesized as precursors, with extra sequences both 5′ and 3′ of the sequences comprising the mature tRNA. A small fraction of tRNAs contain introns. As with rRNA-encoding genes, tRNA-encoding genes are also vigorously transcribed.

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مواضيع ذات صلة

Phosphorylation Activates Pol II

Formation of the Pol II Transcription Complex

Eukaryotic Promoters Are More Complex

Bacterial Promoters Are Relatively Simple

RNA Synthesis is A Cyclical Process that Involves RNA Chain Initiation, Elongation, & Termination

RNA is Synthesized From A DNA Template by RNA Polymerases

DNA & Chromosome Integrity Is Monitored Throughout the Cell Cycle

All Organisms Contain Elaborate Evolutionarily Conserved Mechanisms to Repair Damaged DNA

Micro-RNA and Long Noncoding RNA

Histone Organization

Noncoding DNA

DNA Synthesis Occurs During the S Phase of the Cell Cycle