Post transcriptional modifications

A primary transcript is a linear copy of a transcriptional unit-The the segment of DNA between specific initiation and termination sequences. The primary transcript of both prokaryotic and eukaryotic t RNA s and r RNA s are post-transcriptionally modified by removing extra nucleotides .t RNA s are then further modified to acquire special characters unique to t RNAs. In fact, many of the tRNA transcription units contain more than one molecule. Thus, in prokaryotes, the processing of these rRNA and tRNA precursor molecules is required for the generation of the mature functional molecules.

In prokaryotic organisms, the primary transcripts of mRNA-encoding genes begin to serve as translation templates even before their transcription has been completed. This is because the site of transcription is not compartmentalized into a 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.

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 or as a component of the translation machinery such as rRNA, 5S RNA, or tRNA or RNA processing machinery, snRNAs. Processing occurs primarily within the nucleus and includes nucleolytic cleavage to smaller molecules and coupled nucleolytic and ligation reactions (splicing of exons). However, the processes of transcription, RNA processing, and even RNA transport from the nucleus are highly coordinated.

Some of the processes involved in the post-transcriptional modifications of the primary transcript of major RNAs are as follows-

A) Ribosomal RNA

In mammalian cells, the three rRNA molecules are transcribed as part of a single large precursor molecule called, Pre ribosomal RNAs.

The precursor is subsequently processed in the nucleolus to provide the RNA component for the ribosome subunits found in the cytoplasm.

The 23S,16S, and 5S ribosomal RNAs of prokaryotes are produced from a single RNA precursor molecule (figure-1) as are the 28S, 18S and 5.8S r RNAs of eukaryotes. Eukaryotic  5S rRNA is synthesized by RNA polymerase III and modified separately.

Figure-1- showing the formation of mature r RNA from pre-ribosomal RNA. Cleavage and trimming are the mechanisms involved, similar modifications are observed in the processing of eukaryotic r RNA.

The pre-ribosomal RNAs are cleaved by ribonucleases to yield intermediate-sized pieces of r RNAs, which are further trimmed to produce the required r RNA species.

The proteins destined to become components of ribosome-associated with the r RNA precursor prior to and during the post-transcriptional modifications.

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 required to synthesize sufficient copies of each type of rRNA to form the 10⁷ ribosomes required for each cell replication. 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 the cell growth rate.

B) Transfer RNA

The tRNA molecules serve as adapter molecules for the translation of mRNA into protein sequences. Both eukaryotic and prokaryotic transfer RNA s are made from longer precursor molecules that must be modified. The basic mechanism involved are as follows-

1) Splicing- An intron must be removed from the anticodon loop (figure-2)

2)Trimming-  The sequences at both the 5′ and 3′ ends of the molecule are trimmed (figure-2).

3) Base modifications

The tRNAs contain many modifications of the standard bases A, U, G, and C, including methylation, reduction, deamination, and rearranged glycosidic bonds. Further modification of the tRNA molecules includes nucleotide alkylations,

4) CCA attachment (figure-2)

The attachment of the characteristic CpCpAOH terminal at the 3′ end of the molecule by the enzyme nucleotidyl transferase is the most important modification.

The 3′ OH of the A ribose is the point of attachment for the specific amino acid that is to enter into the polymerization reaction of protein synthesis.

The methylation of mammalian tRNA precursors probably occurs in the nucleus, whereas the cleavage and attachment of CpCpAOH are cytoplasmic functions.

Enzymes within the cytoplasm of mammalian cells are required for the attachment of amino acids to the CpCpAOH residues.

Figure-2-The extra nucleotides at both 5′ and 3′ ends of t RNA are removed, an intron from the anticodon arm is removed, bases are modified (not shown here) and CCA arm is attached to form the mature functional t RNA.

C) Eukaryotic m RNA

The RNA molecule synthesized by RNA polymerase II (the Primary transcript) contains the sequences that are found in cytosolic m RNA. The collection of all the precursor molecules for the m RNA is known as heterogeneous nuclear RNA(hn RNA). The primary transcripts are extensively modified in the nucleus after transcription. These modifications include-

a) 5′ Capping

Mammalian mRNA molecules contain a 7-methylguanosine cap structure at their 5′ terminal. The cap structure is added to the 5′ end of the newly transcribed mRNA precursor in the nucleus prior to the transport of the mRNA molecule to the cytoplasm. The 5‘ cap of the RNA transcript is required both for efficient translation initiation and protection of the 5′ end of mRNA from attack by 5-‘3’ exonucleases.

Eukaryotic m RNAs lacking the cap are not efficiently translated.

The addition of the Guanosine triphosphate (Figure-3) part of the cap is catalyzed by the nuclear enzyme guanylyltransferase. Methylation of the terminal guanine occurs in the cytoplasm. and is catalyzed by guanine-7-methyl transferase.

S-Adenosyl methionine is the methyl group donor. Additional methylation steps may occur.

The secondary methylations of mRNA molecules, those on the 2′-hydroxy and the N⁶ of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm.

Figure-3-showing the attachment of 7 methyl guanosine triphosphate at the 5′ end of the primary transcript by a special 5′-5′ linkage. Additionally, methylation can take place at the 2′ OH group of ribose residues of the first two nucleotides.

b) Addition of poly-A tail

Poly(A) tails are added to the 3′ end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence (figure-4). 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 a poly-A tail.

Figure-4- showing the process of polyadenylation of the primary transcript.

 After the m RNA enters the cytosol, the poly-A tail is gradually shortened.

c) Removal of introns  (splicing)

Introns or intervening sequences are the RNA sequences that 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 steps of splicing are as follows-

i) Role of small nuclear RNA (sn RNA)  and Spliceosome

The molecular machine that accomplishes the task of splicing is known as the spliceosome. Spliceosomes consist of the primary transcript, five small nuclear RNAs (U1, U2, U5, U4, and U6) and more than 60 proteins. Collectively, these form a small ribonucleoprotein (snRNP) complex, sometimes called a “snurp” (snRNPs) (Figure-5).Snurps are thought to position the RNA segments for the necessary splicing reactions. These facilitate the splicing of exon segments by forming base pairs with the consensus sequence at each end of the intron. 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 exon-intron (splice) junctions and at the branch site, which is located 20–40 nucleotides upstream from the 3′ splice site.

Figure-5- Showing spliceosome assembly at the splice site.  

ii) Mechanism of excision of introns

The binding of snRNPs brings the sequences of the neighboring exons into the correct alignment for splicing. The 2′-OH group of an adenosine (A) residue (known as the branch site) in the intron attacks and forms a phosphodiester bond with the phosphate at the 5′ end of the intron 1. The newly- feed 3’OH of the upstream exon 1 then forms a phosphodiester bond with the 5’end of the downstream exon 2. The excised intron is released as a “lariat” structure, which is degraded (Figure-6).

Figure-6- showing the process of splicing.

After the removal of all the introns, the mature m RNA molecules leave the nucleus by passing into the cytosol through pores into the nuclear membrane.

Clinical significance

1) Antibodies against snRNPs

In systemic Lupus Erythematosus (SLE), an autoimmune disease, the antibodies are produced against host proteins, including sn RNPs.

2) Mutations at the splice site

Mutations at the splice site can lead to improper splicing and the production of aberrant proteins. For example, some cases of Beta-thalassemia are as a result of incorrect splicing of beta-globin m RNA due to mutation at the splice site.

Biological significance

Alternative Splicing

Alternative patterns of RNA splicing is adapted for the synthesis of tissue-specific proteins.

The pre-m RNA molecules from some genes can be spliced in two or more alternative ways in different tissues. This produces multiple variations of the m RNA and thus diverse set of proteins can be synthesized from a given set of genes (Figure-7).

For example- Tissue-specific tropomyosins are produced from the same primary transcript by alternative splicing. Alternative splicing and processing result in the formation of seven unique α -tropomyosin mRNAs in seven different tissues.

Figure-7- showing the mechanism of alternative splicing.

 Similarly, the use of alternative termination-cleavage-polyadenylation sites also results in mRNA variability (Figure-8). Alternative polyadenylation sites in the immunoglobulin heavy chain primary transcript result in mRNAs that are either 2700 bases long (_m) or 2400 bases long (_s). This results in a different carboxyl-terminal region of the encoded proteins such that the _m protein remains attached to the membrane of the B lymphocyte and the _s immunoglobulin is secreted.

 

Figure-8- showing the mechanism of alternative poly-A sites selection to produce variability in mRNA and thus different proteins can be synthesized from a given set of genes

The general process of transcription can be applied to both prokaryotic cells and eukaryotic cells. The basic biochemistry for each is the same; however, the specific mechanisms and regulation of transcription differ between prokaryotes and eukaryotes.