Lecture 13 - RNA processing Flashcards
How are RNAs processed.
RNA’s often need to be processed before they are functional (to produce proteins).
* Addition of a 5’-cap (capping)
* Addition of a 3’-tail (Polyadenylation)
* Splicing (usually)
Editing (sometimes)
Describe RNA cleavage.
- rRNA and tRNA are usually synthesised as long precursor RNA molecules
- They are processed to the correct length be ribonucleases.
- This ensures that these TNAs are available in the same amounts within the cell.
In E. coli rRNA and several tRNAs are transcribed as one long RNA molecule
Similarly, in S. cerevisiae (eukaryote) three rRNA components are transcribed as one long RNA
Describe cap structure at the 5’ end of eukaryotic mRNA.
- A guanine nucleotide is added to the 5’ end of an mRNA via a 5’-5’ linkage.
- The guanine is subsequently methylated at the N7 position
In higher eukaryotes, 2’ O position of the second (and sometimes that of the third)ribose are also methylated.
- The guanine is subsequently methylated at the N7 position
What are the functions of the 5’ cap.
- Protection of mRNA from degradation
- Translatability - presence of the cap stimulates translation of mRNA
- Transport from the nucleus to the cytoplasm.
Describe the steps involved in capping.
Capping occurs after the first 20-30 nucleotides have been synthesised during transcription.
1. An RNA triphosphatase removes the terminal phosphates at the 5’ end
2. Guanylyl transferase uses GTP to attack GMP
3. The guanine is methylated by a methyltransferase
In some eukaryotes each step is carried out by a different enzyme but in mammals and other Metazoa (1) and (2) are carried out by one enzyme.
Why is polyadenylation important ot transcription
All eukaryotic mRNAs have a 3–polyadenosine tail, consisting of approximately 200 adenosines. It is added post-transcriptionally.
The mRNA is cleaved immediately after a CA between a AAUAAA hexametric sequence and a U or GU rich region
The 3’ end then has adenosines added by poly(A)polymerase.
The exception are mRNAs encoding metazoan histones which have a different structure at eh 3’ end.
What is splicing?
- The initial transcript (precursor mRNA) often includes intron sequences that must be removed to produce the mature mRNA used as a template for translation.
- Introns are found far more frequently in eukaryotes than in prokaryotes.
- Introns are removed by transesterification reactions. In Metazoa most introns are removed by the spliceosome, although there are some that are self-splicing.
The spliceosome is comprised of proteins and snRNAs.
Describe the basic mechnism of splicing.
Splicing is the process of removing non-coding regions called introns from a pre-mRNA molecule to produce mature mRNA, which contains only coding regions known as exons.
There are two main mechanisms of splicing:
Nucleophilic attack: The intron is excised from the pre-mRNA by a nucleophilic attack mechanism.
Lariat structure: The intron is excised as a lariat (loop) structure following two consecutive transesterification reactions.
How are splice sites recognised.
Intron boundaries are marked by specific sequences known as splice sites. These splice sites contain a 5’-GU at the beginning of the intron and a 3’-AG at the end.
The spliceosome, a complex composed of smaller nuclear ribonucleoproteins (snRNPs) containing snRNA and proteins, recognizes these splice sites and facilitates the splicing process.
What is the Exon Junction Complex?
After splicing, an exon junction complex (EJC) is left at the splice junctions.
The EJC serves several functions:
It marks the mRNA as processed.
It interacts with export and translation proteins.
It prevents incompletely processed RNA from being exported.
What is alternative splicing
Alternative splicing allows for the production of different mRNA isoforms from a single gene by selectively including or excluding exons.
This process is essential for generating protein diversity in eukaryotic organisms.
It can be regulated in various ways, including by developmental cues or tissue-specific factors.
What is the function of RNA pol II
RNA pol II is responsible for transcribing the pre-mRNA from DNA.
During transcription initiation, the C-terminal domain (CTD) of RNA pol II becomes phosphorylated.
This phosphorylation recruits enzymes that add a 5’ cap to the RNA, which protects the mRNA and facilitates translation.
As RNA pol II continues elongation, the CTD undergoes further phosphorylation, facilitating recruitment of the splicing machinery to remove introns.
Additionally, the increased phosphorylation recruits the complex responsible for cleaving and adding a poly(A) tail to the 3’ end of the mRNA, which also aids in mRNA stability and translation.
What is RNA editing?
Process that adds/deletes bases from pre-mRNA of chemically alters bases, resulting in an mRNA with bases that don’t match its DNA sequence.
One of the most of the most extreme examples of RNA editing is seen in the mitochondria of trypanosomes.
How do trypanosomes edit RNA?
Insertion:
* Endonucleases cuts mRNA at mismatch
* Uridylyl transferase adds U’s to 3’ end of 5’ mRNA fragment, guided by the gRNA
* RNA ligase ligates the two mRNA fragments
Deletion:
* Endonuclease cuts mRNA at mismatch
* 3-5’ exonuclease removes the bulged base in in mRNA
RNA ligase ligates the two mRNA fragments
Give an exapmle of a gene that has substitution editing.
APOB gene contains 29 exons (separated by 28 introns)
* The exons contain a total of 4,564 coons
* Codon 2,153 is CAA, which is a codon for the amino acid glutamine.
Liver cells - apolipoprotein B-100 protein contains 4,563 amino acids
Intestinal cells - an additional step of pre-mRNA processing occurs: the deamination of the C nucleotide in Codon 2,153 forming apolipoprotein B-48 protein containing 2152 amino acids.
