STRUCTURE OF GLOBIN mRNA

5'-capping

• Capping is the first modification made to RNA polymerase II-transcribed RNA and takes place co-transcriptionally in the nucleus as soon as the first 25-30 nts are incorporated into the nascent transcript.

• Three enzymatic activities are required to generate the cap 0 structure

1. RNA triphosphatase

2. RNA guanylyltransferase

3. Guanine-N7 methyltransferase.

• The initial steps in RNA capping are catalyzed by a dimeric capping enzyme, which associates with the phosphorylated carboxyl-terminal tall domain (CTD) of RNA polymerase II.

• RNA triphosphatase removes the-phosphate from the 5' end of the nascent RNA. The enzyme RNA guanylyltransferase transfers the GMP moiety from GTP to the 5'-diphosphate of the nascent transcript, creating the guanosine 5-5-triphosphate structure.

• In the final step, guanine-N7 methyltransferase transfers methyl groups from S-adenosylmethionine to the N' position of the guanine at the 5' end of the nascent RNA. If mRNA has a methyl group-on. N' position of guanine at the 5' end, then it is called cap 0. This is the first methylation step and occurs in all eukaryotes. In some higher eukaryotes, methyl group addition also occurs at second base.

• But this happens only when the position is occupied by adenine; the reaction involves addition at the N' position.

• In some species, a methyl group is added to the second as well as third nucleoside of the capped mRNA.

• mRNA with methyl groups on the N' position of the guanine and the 2'-OH position of the second nucleotide at the 5' end is known as cap 1.

• This is the predominant cap in multicellular organisms. Similarly, if methyl group is present at both second and third nucleoside then it is known as cap 2.

FUNCTIONS OF 5’-cap

• The 5'-cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as

1. pre-mRNA processing

2. protecting mRNA from degradation

3. nuclear export

4. cap-dependent protein synthesis

• It was previously thought that 5'-capping occurs exclusively in the nucleus, but RNA capping has been noticed in the cytoplasm of mammalian cells and Trypanosomes.

Polyadenylation

Polyadenylation Process:

1. Polyadenylation Overview

Polyadenylation is one of the processes that is carried out in almost all eukaryotic mRNAs, whereby a poly-A tail consisting of approximately 200-250 residues of adenosine are added to the 3' end of the mRNA. The reason for the poly-A tail is primarily to stabilize the mRNA and facilitate the process of nuclear export, with the main aim of ensuring efficient translation of the protein.

2. Signal for Polyadenylation

It begins with the recognition of the signal for polyadenylation, commonly AAUAAA, located between 10 to 30 nucleotides up-stream of the cleavage site. Down-stream from this it also usually includes a GU-rich or U-rich region that is also critical to the process.

3. Cleavage and enzymes involved

Pre-mRNA is cleaved at a position adjacent to the polyadenylation site, preceding the attachment of a poly-A tail. The two protein complexes concerned with this process are Cleavage Stimulation Factor (CstF) and Cleavage and Polyadenylation Specificity Factor (CPSF). After the pre-mRNA has been cleaved, it is handed over to Poly(A) Polymerase (PAP), which adds the poly-A tail to the 3' end.

4. Single vs Two Step Poly-A Tail Addication

There are two rounds of polyadenylation. In the first round, an oligo(A) tail of about 10 residues is added to it. A short tail of about 200 adenosine residues may be further elongated using PABP that binds to the tail and continues this process.

5. Template Independent Process

Unlike transcription whose mRNA synthesis is template-dependent on a DNA, the addition of the poly-A tail represents a template-independent process. This occurs through the action of what is referred to as Poly(A) Polymerase that adds adenosine residues to the tail of the mRNA without any specific nucleotide sequence necessity.

6. The poly-A tail has numerous important functions. First, this tail would stabilize the mRNA and this would result in slowing the degradation of the mRNA. Another function is that it provides an easier transport of mRNA both in the cytoplasm and the nucleus, thus ensuring high rates of translation. Lastly, this functions by attracting more ribosomes to the mRNA, which enhances translation.

7. Cytoplasmic Polyadenylation

Not confined to the nucleus; it can also be performed in the cytoplasm. This is very important during oocyte and early embryo maturation for the translation of specific types of mRNA. The accomplishment of cytoplasmic polyadenylation is through Cytoplasmic Polyadenylation Element Binding Proteins, which are attached to specific sequences within the mRNA.

8. Role during Oocyte Maturation

It's only during oocyte maturation that the poly-A tail of a few mRNAs becomes elongated hence turning these on for translation. This coincidentally is relevant during development since the proteins will be expressed in the right amounts and at the proper time.

9.Alternative Polyadenylation

While there are instances where some mRNAs have more than one polyadenylation site, this typically leads to different mRNA variants because the poly-A tail varies in length. Known as alternative polyadenylation, it allows a single gene to consist of numerous transcripts, and hence protein isoforms. Indeed, the vast majority of mammalian genes are alternatively polyadenylated, and this is one mechanism by which gene expression is regulated because more than 70 percent of genes undergo alternative polyadenylation.

10. Representation in Immunoglobulin mRNAs

Here is a good representation of alternative polyadenylation in immunoglobulin mRNA which has generated the differential in polyadenylation sites within the same mRNA to create different 3' ends transcripts. This has been illustrated to be important for the various antibody molecules produced as a response to infections by the immune system.

Polyadenylation in Bacteria

Another role in bacteria polyadenylation also has. Though, in bacteria, it marks the mRNA for degradation and not for stabilizing the mRNA, while in addition of poly-A tails which is catalyzed by Poly(A) Polymerase I (PAP I) within bacteria; through these, enzymes such as RNase E and RNase G recognize and degrade the mRNA after polyadenylating. This mechanism makes bacteria capable of degrading unwanted or defective mRNA molecules very rapidly.