Post-transcriptional Modification

Primary transcript made by RNA polymerase normally undergoes further alteration, known as post transcriptional processing or modification. In Prokaryotes, mRNA transcribed directly from DNA template and used immediately in protein synthesis while in Eukaryotes, the primary transcript (hnRNA) must be processed to produce the mRNA (active form).

Post-transcriptional modification Definition

Post-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an primary RNA transcript is chemically altered following transcription from a gene to produce a mature, functional RNA molecule that can then leave the nucleus and perform any of a variety of different functions in the cell.

Post-transcriptional modification also known as the co-transcriptional modification.
Post transcriptional modifications are also responsible for changes in rRNA, tRNA and other special RNA like srpRNA, snRNA, snoRNA, miRNA etc.

Post-transcriptional modification mainly occurs in nucleus of cell.
There are 3 major stepsinvolve in Post-transcriptional modification that significantly modify the chemical structure of the RNA molecule: the addition of a 5′ cap, the addition of a 3′ polyadenylated tail, and RNA splicing.
The splicing helps to remove the introns and links the exons directly, while the cap and tail facilitate the transport of the mRNA to a ribosome and protect it from molecular degradation.

Play a crucial role in generating the heterogeneity in proteins.
Help in utilizing identical proteins for different cellular functions in different cell types.
Regulation of particular protein sequence behavior in most eukaryotic organisms.

Play an important part in modifying the end product of expression.
Contribute towards biological processes and diseased conditions.
Translocation of proteins across biological membranes.

Importance of Post-transcriptional modifications

Modifications help the RNA molecule to be recognized by molecules that mediate RNA translation into proteins.
During post-transcriptional processing, portions of the RNA chain that are not supposed to be translated into proteins are cut out of the sequence. In this way, post-transcriptional processing helps increase the efficiency of protein synthesis by allowing only specific protein- coding RNA to go on to be translated.
Without post-transcriptional processing, protein synthesis could be significantly slowed, since it would take longer for translation machinery to recognize RNA molecules and significantly more RNA would have to be unnecessarily translated.

Post-transcriptional modification Steps

The following modifications occur during the Post-transcriptional modification of proteins;

Addition of 5′ cap

The ends of eukaryotic transcript are processed. Modified guanosine, 7- methyl guanosine is added to the 5′ end in an uncommon 5′-to-5′ (instead of 3′-to-5′) linkage; this terminal group is known as a cap.
After completion of transcription, the 5′ end of the RNA transcript carries a free triphosphate group since it was the first incorporated nucleotide in the chain. The capping process replaces the triphosphate group with another structure called the “cap”. The cap is added by the enzyme guanyl transferase. This enzyme catalyzes the reaction between the 5′ end of the RNA transcript and a guanine triphosphate (GTP) molecule.

During the reaction, the beta phosphate of the RNA transcript displaces a pyrophosphate group at the 5′ position of the GTP molecule. The cap is formed through a 5′-5′ linkage between the two substrates .
Capping protects the 5’ from enzymatic degradation in the nucleus and assists in export to the cytosol.
Eukaryotic m RNAs lacking the cap are not efficiently translated.

Process of 5′ capping

This capping process involves the addition of an extra nucleotide at the 5′ end of the mRNA and methylation by the addition of a methyl group (CH3) to the base in the newly added neucleotide and (may be) to the 2’–OH group of the sugar of one or more nucleotides at the 5′ end.

Importance of 5’ capping

It is necessary for the mRNA to bind with the ribosome to begin protein synthesis (Cap binding proteins first identify the cap and attach to it; a ribosome then binds to these proteins and moves downstream along the mRNA until the start codon is reached and translation begins).

Its presence also increases the stability of mRNA and influences the removal of introns.

The Addition of the Poly A tail

The 3′ terminus of a eukaryotic mRNA molecule is transformed by the addition of a polyadenosine sequence (the poly-A tail) of as many as 50 to 250 nucleotides. These nucleotides are not encoded in the DNA but are added after transcription in a process known as poly-adenylation.
Post-transcriptional RNA processing at the opposite end of the transcript comes in the form of a string of adenine bases attached to the end of the synthesized RNA chain.

The addition of the adenines is catalyzed by the enzyme poly (A) polymerase.
The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence.
Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues.

The poly(A) tail appears to protect the 3′ end of mRNA from 3′ 5′ exonuclease attack.
Histone and interferon’s mRNAs lack poly A tail.
After the m-RNA enters the cytosol, the poly A tail is gradually shortened.

Process of addition

Processing of the 3′ end of pre-mRNA requires sequences both upstream and downstream of the cleavage site.
The consensus sequence AAUAAA is usually from 11 to 30 nucleotides upstream of the cleavage site and determines the point at which cleavage will take place.
A sequence rich in Us (or Gs and Us) is typically downstream of the cleavage site.

At the cleavage site, as many as 250 nucleotides are added following a complex process with the help some factors.

Importance of Poly A tail

The poly(A) tail confers stability on many mRNAs, increasing the time during which the mRNA remains intact and available for translation before it is degraded by cellular enzymes.

RNA Splicing

RNA splicing is the process by which introns, regions of RNA that do not code for proteins, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule.

Splicing occurs in the nucleus following transcription but before the RNA moves to the cytoplasm.
RNA splicing takes place in nuclear particles known as spliceosomes. These abundant particles are composed of protein and several types of specialized small nuclear RNA (snRNA) molecules ranging from 100 to 200 bases in length.
Introns or intervening sequences are the RNA sequences which do not code for the proteins.

These introns are removed from the primary transcript in the nucleus, exons (coding sequences) are ligated to form the mRNA molecule, and the mRNA molecule is transported to the cytoplasm.
The molecular machine that accomplishes the task of splicing is known as the spliceosome.
Small nuclear RNA molecules that recognize splice sites in the pre- mRNA sequence.

The excised intron is released as a “lariat” structure, which is degraded.
The exons portion of mRNA translated into a protein. These are the coding portions of a mRNA molecule.
RNA splicing mainly occurs after the complete synthesis and end-capping of the pre-mRNA, transcripts with many exons can be spliced co-transcriptionally.

This reaction is catalyzed by a large protein complex known as spliceosome, which is consists of proteins and small nuclear RNA molecules that recognize splice sites in the pre-mRNA sequence.
Many pre-mRNAs, including those encoding antibodies, can be spliced in multiple ways to create different mature mRNAs that encode different protein sequences. This process is known as alternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.

Alternative Splicing

Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins.

In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. This means the exons are joined in different combinations, leading to different (alternative) mRNA strands.
The proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions.
Alternative patterns of RNA splicing is adapted for the synthesis of tissue-specific proteins.(antibodies).

The pre-mRNA molecules from some genes can be spliced in two or more alternative ways in different tissues.
This produces multiple variations of the mRNA and thus diverse sets of proteins can be synthesized from a given set of genes.
Alternative splicing occurs as a normal phenomenon in eukaryotes.

Introns are removed from the primary transcript in the nucleus, exons (coding sequences) are ligated to form the mRNA molecule. (After removal of all the introns, the mature m RNA molecules leave the nucleus by passing in to the cytosol through pores in to the nuclear membrane).
Alternative splicing accomplishes this in two ways: a) by splicing together exons from two different primary RNA transcripts in a process called trans- splicing b) by splicing out entire exons.

Alternative Splicing | Source: https://en.wikipedia.org/wiki/Alternative_splicing#/media/File:DNA_alternative_splicing.gif

Covalent modifications

The proteins synthesized in translation are subjected to many covalent changes. By these modifications in the amino acids, the proteins may be converted to active form or inactive form. Selected examples of covalent modifications are described below.

Phosphorylation

It involves the addition of a phosphate (PO4) group to a protein or a small molecule.
The hydroxyl group containing amino acids of proteins, namely serine, threonine and tyrosine are subjected to phosphorylation.
The phosphorylation may either increase or decrease the activity of the proteins.

A group of enzymes called protein kinases catalyse phosphorylation while protein phosphatases are responsible for dephosphorylation (removal of phosphate group).
Many enzymes that undergo phosphorylation or dephosphorylation are known in metabolisms (e.g. glycogen synthase).

Hydroxylation

It involves the addition of hydroxyl group to proline of protein.

During the formation of collagen, the amino acids proline and lysine are respectively converted to hydroxyproline and hydroxylysine. This hydroxylation occurs in the endoplasmic reticulum and requires vitamin C.

Glycosylation

It involves the addition of saccharide to a protein or a lipid molecule.
The complex carbohydrate moiety is attached to the amino acids, serine and threonine (O-linked) or to asparagine (N-linked), leading to the synthesis of glycoproteins

Biotinylation

It involves the addition of biotin to protein or nucleic acid.

Acetylation

It involves the addition of an acetyl group, usually at the N- terminus of the protein.

Carboxylation

It involves the addition of carboxyl group to glutamate.

Methylation

The addition of a methyl group, usually at lysine or arginine residues.

Alkylation

The addition of an alkyl group (e.g. methyl, ethyl).

Glutamylation

Covalent linkage of glutamic acid residues to tubulin and some other

Lipoylation

The attachment of a lipoate functionality Sulfation The addition of a sulfate group to a tyrosine.

Attachment of Carbohydrate Side Chains

The carbohydrate side chains of glycoproteins are attached covalently during or after synthesis of the polypeptide.
In some glycoproteins, the carbohydrate side chain is attached enzymatically to Asn residues (N-linked oligosaccharides), in others to Ser or Thr residues (O-linked oligosaccharides).

Many proteins that function extracellularly, as well as the lubricating proteoglycans that coat mucous membranes, contain oligosaccharide side chains.

Addition of Isoprenyl Groups

A number of eukaryotic proteins are modified by the addition of groups derived from isoprene (isoprenyl groups).
A thioether bond is formed between the isoprenyl group and a Cys residue of the protein.

The isoprenyl groups are derived from pyrophosphorylated intermediates of the cholesterol biosynthetic pathway, such as farnesyl pyrophosphate.
Proteins modified in this way include the Ras proteins, products of the ros oncogenes and proto-oncogenes, and G proteins, and Iamins, proteins found in the nuclear matrix.
The isoprenyl group helps to anchor the protein in a membrane.

Addition of Prosthetie Groups

Many proteins require for their activity covalently bound prosthetic groups.
Two examples are the biotin molecule of acetylCoA carboxylase and the heme group of hemoglobin or cytochrome c.

Proteolytic Processing

Many proteins are initially synthesized as large, inactive precursor polypeptides that are proteolytically trimmed to form their smaller, active forms.

Examples include proinsulin, some viral proteins, and proteases such as chymotrypsinogen and trypsinoge.

Formation of Disulfide Cross-Links

After folding into their native conformations, some proteins form intrachain or interchain disulfide bridges between Cys residues.
In eukaryotes, disulfide bonds are common in proteins to be exported from cells.

The cross-Iinks formed in this way help to protect the native conformation of the protein molecule from denaturation in the extracellular environment, which can differ greatly from intracellular conditions and is generally oxidizing.

References

Principles of Genetics, D. Peter Snustad, Michael J. Simmons.
Biochemistry, Dr. U. Satyanarayana, Dr. U. Chakrapani.

https://en.wikipedia.org/wiki/Post-transcriptional_modification
https://www.basu.org.in/wp-content/uploads/2021/06/Post-Transcriptional-Modifications_1.pdf
https://www.slideshare.net/sadiqpa/post-transcriptional-modification

https://www.slideshare.net/AYSHA007/posttranslational-modifications
https://www.slideshare.net/saadiaeman/post20translational20modifications
https://www.slideshare.net/sujay45/posttranslational-modification

https://plantlet.org/post-transcriptional-modification/
https://en.wikipedia.org/wiki/Alternative_splicing