Transcription & RNA Processing 2 (mRNA)

Question 1

How does protein synthesis occur in prokaryotes?

What is this process called?

Answer 1

1. In prokaryotes, protein synthesis occurs immediately after a gene is transcribed during transcription.
2. This is called coupling.

Question 2

Why does mRNA have to be processed before protein synthesis can occur?

Answer 2

Because of the nuclear envelope, mRNA must be modified through several forms of processing and then transported to the cytosol where ribosomes can start protein synthesis.

Question 3

What forms of processing does mRNA undergo?

Answer 3

mRNA processing includes:
Capping: modification of 5’ end with 7-methylguanosine
Tailing: polyadenylation of 3’ end
Splicing: removal of introns or joining of exons
and sometimes RNA editing

Question 4

What is the process of mRNA capping?

What are the functions of capping?

Answer 4

1. After transcription of the first 20 to 30 bases of RNA, a GTP is added in reverse to the 5’ end of pre-mRNA then methyl groups are added to the G and to the riboses of 1 or 2 of the next bases in the chain.
2. The 7-methylguanosine cap on the 5’ end of mRNA prevents it from being degraded by RNAses that splice out intronic RNA from the 5’ end.
   It also serves as a binding site that helps the mRNA align with the ribosome.

Question 5

What is mRNA tailing?
What is the poly AAA site?
What is the process of mRNA tailing?

Answer 5

1. In a reaction called polyadenylation, a poly A tail is added to the 3’ end of the pre-mRNA.
2. The poly AAA site is where the tail is added. There is an AAUAAA signal 10 to 30 bases upstream of the site and a GU rich element downstream (although some have one upstream) of the site.
3. Attracted to the phosphorylated CTD of RNA polymerase 2, an endonuclease cleaves the downstream element and a polyA-polymerase adds a poly A tail of 200 bases. The downstream element is degraded into nucleotides, leading to termination of translation.

Question 6

What are the functions of mRNA tailing?

What is the best example of mRNA tailing?

Answer 6

1. Poly A tails can regulate translational efficiency (ribosome recognition) and mRNA stability (keeps the 3’ end from being degraded).
2. Best example are maternal mRNA’s which are stored with short tails that become lengthened after fertilization.

Question 7

What is mRNA splicing?

Answer 7

The pre-mRNA transcript has 5’ and 3’ untranslated regions (UTRs) as well as protein-coding segments (exons) and code-interrupting segments (introns). Splicing is the pre-mRNA processing step in which introns are removed and exons are linked together.

Question 8

What is the process of mRNA splicing?

Answer 8

Sequences in the RNA indicate the splice sites by marking the end of an exon and beginning of an intron.

1. Cleavage at the 5’ splice site leaves an exon with a free 3’ end. The 5’ end of the intron is joined to a 2’ hydroxyl of an adenine base that is near the 3’ end of the intron. This forms a loop and turns the entire intron into a lariat-like structure.
2. The 3’ end of the intron is cleaved to release the entire intron lariat and the free 3’ end of the first exon is joined to the now free 5’ end of the next exon.

Question 9

What is the spliceosome?

Answer 9

Five snRNAs (U1, U2, U4, U5, U6) each bind 6 to 10 proteins to form small nuclear ribonucleoprotein particles (snRNPs). These snRNPs combine to form the spliceosome.

Question 10

How does the spliceosome participate in splicing?

Answer 10

1. ) U1snRNP binds the 5’ splice site, which then allows U2snRNP to bind an internal site within intron. This bends the intron and brings the first exon closer to the second.
2. ) U4/U6snRNP and U5snRNP preformed complex enters the spliceosome.
3. ) U5snRNP binds to sequences upstream of the 5’ splice site, so that U5snRNP is holding the first exon.
4. ) U4snRNP leaves the complex, also displacing the U1snRNP.
5. ) U6snRNP forms a complex with U2snRNP and two things occur: The exon is cleaved from the intron at the 5’ splice site and the 3’ end of the intron is joined to its internal adenine, catalyzing formation of the 2’ hydroxyl link and creating the lariat.
6. ) The U5snRNP is left holding a free exon positioned above the 3’ splice site
7. ) U5snRNP binds the 3’ splice site leading to the cleavage and removal of the lariat intron and the linkage of the first and second exons.

Question 11

What other two protein factors are involved in targeting the splicing reaction?

Answer 11

1. SR proteins can bind to sites in exons and recruit the snRNP’s to initiate spliceosome assembly.
2. U2AF binds to the 3’ sites and recruits U2 to the branch point within the intron.

Question 12

Why are the other two protein factors important in targeting the splicing reaction?

Answer 12

SR proteins and U2AF bind the CTD of RNA polymerase 2 and thereby maintain the correct order of splicing by coupling it with transcription.
They also mediate alternative splicing.

Question 13

What two snRNAs demonstrate self-splicing ability?

Answer 13

U2 and U6 snRNAs have been shown capable of catalyzing both steps of the splicing reaction on their own (in the absence of proteins).

Question 14

What is alternative splicing?

What is the benefit of alternative splicing?

Answer 14

1. Given the process of splicing, there then exists the potential, through varying the order or inclusion of exons within an RNA message, of altering the resultant proteins amino acid sequence.
2. This increases the diversity of the number of proteins that are encoded within the DNA.

Question 15

What is the process of alternative splicing?

Answer 15

Alternative splicing is usually accomplished by non-spliceosome factors that act as activators and recruit the spliceosome snRNPs to specific alternative sites.
Some factors can also act as repressors and block the ability of snRNPs to bind to certain splicing sites

Question 16

What is RNA editing?

Answer 16

RNA editing is the process of changing single bases in RNA to alter the amino acid sequence of the protein product.

Question 17

What is an example of RNA editing in the small intestine?

Answer 17

Apolipoprotein B (Apo-B) mRNA is edited to produce two distinct proteins in the liver and the small intestine. In the liver, the full length protein Apo-B100 is produced (4536aa). In the small intestine, the message for B-100 is edited by deamination of cytosine to uracil to create a stop codon (CAA UAA) roughly halfway through the message, yielding Apo-B48 (2152aa).

Question 18

What is an example of RNA editing in neuronal receptors?

Answer 18

Deamination of adenosine to inosine plays a role in neuronal receptors, where the base change yields an amino acid substitution. In serotonin, such changes can occur in 5 sites yielding 24 different versions of the receptor with potentially different properties.
Mutation of such editing in model organisms yields phenotypes with neurological defects.

Question 19

How is pre-mRNA degraded?

Answer 19

Over 90% of pre-mRNA sequence is introns and thus needs to be degraded:

1. ) The lariat structures are targeted specifically by enzymes recognizing the 2’-5’ bond.
2. ) The 3’ ends and 5’ ends of RNA are also targeted, as mature mRNA is capped or tailed.
3. ) When ribosomes encounter premature termination codons, the mRNA may be degraded by nonsense mediated mRNA decay.

Question 20

How can the degradation rate of normal mRNAs represent a control mechanism for protein expression?

Answer 20

Rates vary from less than 30 minutes to greater than 20 hours for different mRNAs

1. ) Shorter half lives are usually assigned to regulatory proteins that rapidly respond.
2. ) Longer half-lives are assigned to structural proteins.
3. ) The length of the poly AAA tail is generally related to the half-life of the mRNA.

Question 21

How are other factors involved in normal mRNA degradation?

Answer 21

1. ) Some factors bind to sequences at the 3’ ends of mRNAs to stabilize them (preventing degradation in the absence of a long AAA tail).
2. ) Some factors bind to sequences in the 5’ end of mRNAs to block translation.
3. ) siRNAs and miRNAs can impact degradation as well.