A process in cell biology by which, primary transcript RNA is converted into mature RNA.

POST-TRANSCRIPTIONAL MODIFICATION

Vital for the correct Translation

RNA transcript contains both Exons and Introns Occurs in the cell nucleus in case of Eukaryotes

THE PRE-RNA MOLECULE UNDERGOES THREE MAIN MODIFICATIONS:

5' CAPPING

3' POLYADENYLATION
RNA SPLICING

AN OVERVIEW

5 PROCESSING

5 CAPPING

Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end. The capping process occurs in nuclei

Guanosine is methylated on the 7 position directly after capping in vivo by a methyl transferase.
This type of cap, with just the (m7G) in position is called a Cap 0 Structure. Provides significant resistance to 5' Ribonucleases.

M7GPPPGP----

OH

OH O N O H2C O P O O O NH N NH 2

H2N HN

O N N

O P O P O O O

CH2 O

5'

5'
O

Pi

O O P O O

OH AAAAA-OH

3'

ppp5'NpNp
Pi

removing phosphate group

pp5'NpNp
GTP PPi

forming 5'-5' triphosphate group

G5'ppp5'NpNp methylating at G7
m
7

GpppNpNp methylating at C2' of the first and second nucleotides after G

Gpppm2'Npm2'Np

CAPPING PROCESS
1.

One of the terminal phosphate groups is removed leaving two terminal phosphates.
(RNA terminal phosphatase)

2.

GTP is added to the terminal phosphates, losing two phosphate groups in the process. This results in the 5' to 5' triphosphate linkage.
(Guanyl transferase)

3.

The 7-nitrogen of guanine is methylated.

(Methyl transferase)

4.

Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides

CAP FUNCTIONS:
The 5' cap has 4 main functions:
1.

Regulation of nuclear export.

Cap binding complex (CBC) binds exclusively to capped RNA Recognized by the Nuclear Pore Complex

2.

Prevention of degradation by Exonucleases.

Functionally look like a 3' end preventing Exonucleases Block the access of decapping enzymes increasing the halflife of the mRNA

3.

Promotion of translation
5' cap is a marker of an actively translating mRNA regulate mRNA half-lives in response to new stimuli Undesirable mRNAs are sent to P-bodies for temporary storage or decapping

4.

Promotion of 5' proximal Intron excision.

5' cap appears to loop around and interact with the spliceosome

3 PROCESSING

3' PROCESSING:
CLEAVAGE AND POLYADENYLATION

Involves cleavage of its 3' end and then the addition of about 200 Adenine residues to form a Poly(A) tail.

As the poly(A) tails is synthesised, it binds multiple copies of poly(A) binding protein, which protects the 3'end from ribonuclease digestion

RNA SPLICING

SPLICING

Process by which Introns, are removed from the pre-mRNA and the remaining Exons connected to re-form a single continuous molecule. The splicing reaction is catalyzed by a large protein complex called the Spliceosome. Some pre-mRNA can be spliced in more than one way, generating alternative mRNAs. This is called alternative Splicing.

THE CHEMISTRY OF RNA SPLICING

5 Splice site & 3 Splice sites. (Sometimes referred to as Donor & Acceptor sites)

Branch Point Site or Branch point Sequence followed by Polypyrimidine tract (Py tract). Highly conserved sequences are the GU in the 5 splice site, the AG in the 3 splice site, and the A at the branch site.

The intron is removed in a form called a Lariat as the Flanking Exons are joined. Two successive Transesterification Reactions. In 1st reaction the 2 -OH of the Conserved A at Branch site acts as nucleophile to attack the Phosphoryl group of the Conserved G in 5 splice site.

In 2nd reaction the 3 -OH of the 5 exon becomes the nucleophile that attacks the Phosphoryl group at the 3 splice site.

Exons from Different RNA Molecules Can Be Fused By Trans-Splicing. In Alternative splicing, exons can be skipped, and a given exon is joined to one further downstream. In some cases, two exons carried on different RNA molecules can be spliced together in a process called trans-splicing.

THE SPLICEOSOME MACHINERY:

RNA splicing is carried out by a large complex called the Spliceosome. Comprises about 150 proteins and 5 RNAs and is similar in size to a ribosome. Several molecule of ATP hydrolyzed.

The 5 RNAs, U1, U2, U4, U5 & U6 ate collectively called Small Nuclear RNAs (snRNAs).

THE ASSEMBLED SPLICEOSOME:

SPLICING:

Each RNA is 100 to 300 nucleotides long and is complexed with proteins.
Small Nuclear Ribonuclear Proteins(snRNPs)

snRNPs have three roles in Splicing.

1st they Recognize the 5 splicing site and the branch site; 2nd they bring those sites together; 3rd they catalyze the RNA cleavage and joining reaction.

Assembly, Rearrangements & Catalysis within Spliceosome

SPLICING PATHWAYS

ASSEMBLY:

5 splice site recognized by U1 snRNP. One subunit of U2AF binds to Py tract & other to 3 splice site. Former interacts with BBP(Early [E] Complex). U2 snRNP binds to branch site(A-Complex). U4, U5 & U6 joins the complex forming BComplex. U1 leaves & U6 replace it at 5 splice site.

CATALYSIS:

U4 is released, U6 interacts with U2 (C-Complex). This facilitates the 1st Transesterification reaction.

U5 aids the 2nd reaction which brings the two exons together.
Finally release the mature RNA and the snRNPs are also released after degrading the Lariat.

POST TRANSCRIPTIONAL MODIFICATION IN EUKARYOTE IS MORE CRITICAL THAN IN PROKARYOTE.

3.2 Modification of tRNA

Cleavage

RNAase P endonuclease

ligase

Addition of CCA-OH

tRNA nucleotidyl transferase

ATP ADP

Base modification
1. Methylation AmA, GmG
2. Reduction UDHU 3. Transversion U
3 4

2 1

4. Deamination AI

R-RNA MODIFICATION:
45S transcript in nucleus is the precursor of 3 kinds of rRNAs.

rRNA
28S

18S

5.8S

45S-rRNA

transcription

splicing
18S-rRNA
5.8S and 28S-rRNA