POST TRANSCRIPTIONAL MODIFICATIONS Complete Process
The flow of genetic information is relatively simple and fast in prokaryotes where both transcription and translation are coupled in the protoplasmic compartment. But in eukaryotes, the nuclear envelope is a hurdle in this smooth flow with transcription taking place in the nuclear compartment and translation in the cytoplasmic compartment. Besides, the product of transcription in eukaryotes is a raw transcript (called primary transcript or pre-RNA) which undergoes various chemical changes to become a functional molecule. The term RNA processing refers to all these chemical modifications necessary to generate a final RNA product from the primary transcript. Processing typically involves the addition or chemical modifications of specific nucleotides and also the removal of certain portions of precursor RNA (Table 5.1). Although processing is more often related to the eukaryotic system, nevertheless prokaryotic rRNAs and tRNAs (not mRNAs) are also synthesized as large precursor molecules which need to be processed to yield mature RNAs. All the three major classes of RNA (mRNA, rRNA, and tRNA) in eukaryotes undergo varying degrees of modifications in the nucleus prior to their export into the cytoplasm where they are utilized for translating the genetic information into a functional product, the protein.
The central dogma (DNA -> RNA -> Protein) as given by Francis Crick in 1956 suggested co-linearity between gene and polypeptide. This means that a gene consists of a continuous sequence of nucleotide pairs, which specify the collinear sequence of amino acids in its polypeptide product [Fig. 1]. This is true for genes found in bacterial cells and many viruses. At first, eukaryotic genes and their polypeptides were also assumed to be colinear. Then in 1997 Alec Jeffreys and Richard Flavell of the University of Amsterdam reported a eukaryotic gene interrupted by a stretch of nucleotides that are not represented in either the functional mRNA or its protein product. They discovered an intervening sequence of approximately 680 bases located directly within a part of the rabbit β-globin gene that coded for an amino acid sequence of the globin polypeptide. Intervening sequences were soon discovered in other genes also and it becomes apparent that the genes with intervening sequences – called split genes are much more common than uninterrupted genes in higher animals and plants. These intervening sequences are called Introns and the sequences that remain present in the mature mRNA are called Exons (expressed sequences).
Splicing mechanisms
The discovery of introns in genes generated interest in finding out the mechanisms by which these intron sequences are prevented from being expressed in the protein product. The demonstration that RNA polymerase transcribes intron sequences along with the exon sequences and ribosomes do not skip any codon while translating the messenger RNA finally focussed the research on the removal of introns from the primary gene transcript. This entire process of removal of introns from the precursor RNA and joining of exons to form functional RNA is called RNA splicing.
There are three distinct mechanisms of intron excision from RNA transcripts.
1. The introns of nuclear pre-mRNAs are spliced out by complex ribonucleoprotein particles called spliceosomes.
2. The introns of some rRNA precursors are removed autocatalytically by the RNA molecule itself.
3. The introns of tRNA precursors are excised by enzymes – splicing endonuclease and splicing ligase.
Spliceosomes remove introns from pre-mRNA
Conserved sequences within introns
How does the splicing machinery recognize the exon-intron junctions in different pre-mRNAs? Analysis of the base sequences of hundreds of different introns ranging from yeast to insects to mammals revealed the presence of conserved sequences at the splice sites. the most commonly found sequences at exon-intron borders within mammalian pre-mRNA. The GU at 5’ splice site and AG at 3’ splice site are the most highly conserved sequences in the vast majority of eukaryotes. Besides, there is a third sequence present within the intron, which is necessary for splicing. This is called branchpoint site (or branch point sequence) having 100% conserved A (adenine nucleotide) followed by a polypyrimidine tract. The base sequence of the remainder of the intron is irrelevant to the process of splicing. Though the sizes of introns vary from tens to thousands of nucleotides, most parts of the intron can be artificially removed without altering the splicing process. But any mutations in the conserved sequences prevent splicing, leading to the accumulation of unspliced precursors. For example, certain types of disorders like inherited thalassemia are commonly caused by mutations in the splice sites of the globin gene.
Chemistry of splicing
Removal of introns requires cuts at both the splice sites so that the flanking exons (5’ exon & 3’ exon) become free to join with each other. This process takes place through two transesterification reactions. In the splicing reactions mentioned above, there is no net gain in the number of chemical bonds – two phosphodiester bonds are broken and two new ones are made. Thus no energy is required for these reactions. a large amount of ATP is consumed during splicing. This energy is actually required to assemble and operate the splicing machinery properly.
Spliceosome machinery
The splicing reactions described above are mediated by huge molecular machinery called the spliceosome, comprising of RNA and proteins. Spliceosomes are in many ways like small ribosomes. Each spliceosome comprises 5 small nuclear RNAs (snRNAs) and about 150 proteins. Because they are rich in uridine these snRNAs are named U1, U2, U4, U5 and U6 (U3 snRNA is located in the nucleolus and is probably involved in the processing of rRNA). These snRNAs, which range in size from 100 to 300 nucleotides in most eukaryotes, are complexed with several protein molecules to form small nuclear ribonucleoprotein particles (snRNPs; sometimes called “snurps”). The U1, U2, and U5 snRNPs each contain a single snRNA molecule whereas U4 and U6 snRNAs are complexed to each other in a single snRNP. Spliceosomes are not present within the nucleus in a prefabricated state rather they are assembled in a stepwise manner as their component snRNPs bind to pre-mRNA. Once the spliceosome machinery is assembled, the snRNPs carry out the reactions that cut the intron out of the transcript and paste the exons together. There are also many proteins within the spliceosome that are not components of snRNP but play critical roles in spliceosome assembly, particularly in the identification of correct splice sites in pre-mRNAs. Introns frequently contain many sequences that resemble splice sites so the splicing machinery must be able to identify the appropriate 5’ and 3’ splice sites at the intron-exon boundaries to produce a functional mRNA. These proteins bind to specific RNA sequences and then recruit snRNPs to the appropriate sites on the intron. One example is U2AF (U2 auxillary factor) which recognizes and binds to the polypyrimidine tract/3’ splice site and recruits U2 snRNA to the branch site. There is another class of proteins called SR proteins (rich in the amino acids serine and arginine) which bind to specific sequences within exons and recruit U1 snRNP to 5’ splice site and U2AF to 3’ splice site. SR proteins along with many other components of spliceosome become associated with CTD (carboxy-terminal domain) of RNA Polymerase II as transcription begins. This anchoring of the splicing machinery to RNA polymerase ensures that exons are joined in the correct order and no exon is skipped during mRNA processing.
Autocatalytic splicing
Some introns are self-splicing meaning that they can catalyze their own removal without the aid of any other protein or RNA molecule. This autocatalytic splicing was first discovered in the Tetrahymena rRNA precursor. self-splicing introns can be grouped into two categories on the basis of their structure and splicing mechanism. They are referred to as Group I and Group II introns. Group I introns are found in a variety of genes, including some rRNA genes in protists, some mitochondrial genes in fungi, mitochondrial and chloroplast genes in plants, and even some bacteriophage genes. Though the nucleotide sequence of Group I introns may be quite variable, all of them share a conserved secondary structure. The key structural features of these introns are – a binding site for guanine nucleoside or nucleotide (guanosine, GMP, GDP, or GTP) and an “internal guide sequence” that base pairs with the 5’ splice site sequence and thereby determine the precise site of cleavage.
The other class of self-splicing introns is Group II introns. They are found in some mitochondrial and chloroplast genes of plants and fungi, and in the genomes of some archaea and bacteria. Like Group I introns they also fold into the characteristic secondary structure. But the chemistry of splicing and RNA intermediates produced is the same as those for spliceosomal-mediated splicing of nuclear genes. They use an adenine residue within the branch site to produce a lariat and finally, the intron is released in the form of a lariat. Because of these similarities, Group II introns and nuclear pre-mRNA introns have been suggested to be evolutionarily related. Perhaps the nuclear pre-mRNA introns evolved from self-splicing group II introns and later snRNAs of the spliceosome took over the catalytic function.
Enzymatic excision of introns in tRNA
The excision of the intron occurs in two steps. In Step I a specific splicing endonuclease recognizes the intron termini and makes a cut at each of the intron-exon junction. The specificity of this reaction resides in the conserved three-dimensional structure of tRNA precursors, not in the nucleotide sequence at splice sites. In step II splicing ligase joins the two exon ends to complete the splicing reaction.
Introns are thought to have played an important role in the evolution of protein-coding genes by facilitating recombination between exons of different genes – a process known as exon shuffling. Exons are shuffled when recombination takes place between different genes. Any recombination event requires break-in genes, which can occur within introns without introducing mutations that might harm the organisms. Moreover, exons tend to be much shorter (approximately 150 nucleotides) than introns (several hundred kilobases). This difference in size ensures that recombination is more likely to occur within the introns than within the exons. The presence of conserved sequences at 5’ and 3’ splice sites allows the splicing machinery to remove introns from recombinant genes in the same manner as in other genes, thereby ensuring the expression of almost all recombinant genes. During the course of evolution, almost an infinite number of new combinations of exons must have occurred for new and useful coding sequences.
RNA Editing
RNA editing is another post-transcriptional modification that alters the base sequence of a eukaryotic mRNA so as to encode a different protein from that encoded by the gene. Two mechanisms of RNA editing are modification of specific bases and the addition or deletion of U residues at multiple sites in RNA. The first type of RNA editing has been well documented in mammalian nuclear mRNAs as well as in mitochondrial and chloroplast RNAs of higher plants and involves site-specific deamination of cytosines and adenines. One of the best-studied examples is the editing of mRNA for apolipoprotein B in the human which transports lipids in the blood. There is a particular CAA codon approximately in the middle of apolipoprotein B mRNA and the C within this codon is deaminated. The deamination is carried out by the enzyme cytidine deaminase which converts C to U. This deamination is tissue specific. In the liver, mRNA is not edited, CAA codes for glutamine and a full-length protein, Apo B-100 (4563 amino acids) is synthesized. Apo B-100, in the liver, is involved in the transport of endogenously synthesized cholesterol and triglycerides. However, in the intestine, apolipoprotein B mRNA is edited converting CAA codon to UAA (stop codon) that prematurely terminates translation resulting in the synthesis of a truncated protein, Apo B-48 (2152 amino acids). This short Apo B-48 protein is involved in the absorption of dietary lipids in the intestine. The cytidine deaminase involved in this editing has an RNA binding domain that helps recognize the specific site for deamination.
Another type of enzymatic deamination is the conversion of Adenosine to Inosine by the enzyme ADAR (adenosine deaminase acting on RNA). Inosine behaves like guanosine in its base-pairing properties and so this change can alter the sequence of the protein encoded by the mRNA. For example, the A to I editing at multiple sites in the mRNAs encoding receptors for the neurotransmitter, serotonin produces different versions of the receptor with different signaling activities. The importance of this type of editing has been demonstrated in the nervous system of Caenorhabditis elegans, Drosophila, and mouse, where a mutation in the editing enzyme leads to various kinds of neurological disorders.
mRNA transport
It has already been discussed that eukaryotic mRNA undergoes various types of processing (capping, splicing, polyadenylation, and editing (in some cases)) before it is ready to be transported to the cytoplasm where it is translated into a polypeptide. Throughout the course of its transition from a primary transcript till it becomes fully mature, an mRNA remains associated with a large number of proteins. Some of these proteins are replaced at various steps along the processing pathways (for example proteins involved in capping, polyadenylation, and splicing) while others remain attached (for example SR proteins) and additional proteins join the RNA (for example a multiprotein complex called exon junction complex is deposited near the boundary of each newly formed exon-exon junction and is required for efficient export of mRNA from the nucleus). Thus, a typical mature mRNA carries a specific set of proteins that marks it for transport. The presence of any alternative combination of proteins with RNA, on the other hand, blocks its export. This mechanism ensures that damaged and unprocessed mRNAs, as well as, excised introns are retained in the nucleus. The transport of mRNA is, thus, mediated by proteins that bind to it. Some of these proteins contain amino acid sequences called nuclear export signals (NES) which target the protein, and hence its bound RNA, for export through the nuclear pores. The nuclear export signals are recognized by transport receptors called exportins that carry this RNA-protein complex out through the pores. The activity of exportin, in turn, is controlled by a GTP-binding protein called Ran. Like other GTP binding proteins, Ran can exist in an active GTP-bound form or an inactive GDP-bound form. Ran-GTP promotes the binding of NES containing ribonucleoprotein (mRNA+proteins) to exportin. Messenger RNA, thus, leaves the nucleus as an RNP-exportin-Ran-GTP complex. Once in the cytoplasm, the GTP is hydrolyzed, the mRNA is released and some of the mRNA binding proteins, RAN-GDP, and exportin shuttle back into the nucleus to escort other mRNAs.
Processing of tRNA
The tRNA molecules in both bacterial and eukaryotic cells are transcribed as precursors (pre- tRNAs) that are extensively modified and processed to produce mature tRNAs. In prokaryotes, several tRNAs are usually transcribed together as one large precursor RNA, which is then cleaved into pieces, each containing a single tRNA. Some tRNAs, in bacteria, are cleaved from the large precursor rRNA. In eukaryotes, tRNA genes are clustered, but RNA polymerase III transcribes each gene individually rather than as a large precursor. Processing of tRNA typically involves the removal, addition, and modification of nucleotides. Pre- tRNAs are generally longer than mature tRNAs and have additional nucleotides at 5’ and 3’ ends called 5’ leader sequence and 3’ trailer sequence respectively. These sequences are removed by specific enzymes. The 5’ leader sequence is cleaved by an enzyme called RNase P which is a special endonuclease consisting of both RNA and protein subunits. In 1983 Sidney Altman and his colleagues demonstrated that RNase P is a ribozyme; its RNA subunit can catalyze the cleavage of pre- tRNA independently of the protein subunit, though both are required for maximal activity. The 3’ trailer sequence is cleaved by a conventional protein RNase.
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