RNA Synthesis and Splicing LO

Question 1

Three major ways in which pre-mRNA are processed

Answer 1

1. Capping
2. Splicing
3. Cleavage/Polyadenylation

Question 2

Capping

Answer 2

Addition of 7-methylGPPP cap to protect the exposed phosphate

Question 3

Splicing

Answer 3

Removal of introns by excision and ligation of exons together to make a complete and readable sequence.

Question 4

Cleavage/ Polyadenylation

Answer 4

Removal of a portion of the 3’ end of the pre-mRNA by cleavage and addition of a polyadenosine tail (important in translation and for stability)

Question 5

Compare and Constrast pre-mRNA and mature mRNA

Answer 5

Pre-mRNA is the primary transcript from DNA. Mature mRNA is ready to be translated while pre-mRNA still needs to be processed before it is ready to be translated. Pre-mRNA contains all the elements of the transcription unit, including introns which must be spliced out (and then exons ligated together). It also lacks a 5’ cap ,and 3’ poly A tail.

Question 6

Functions of 5’ cap of mRNA

Answer 6

Cap Binding Protein:

• regulate splicing by promoting excision of the intron proximal to the cap,
• signal for export to cytoplasm, where CBP is exchanged for eIF4E (eukaryotic initiation factor 4E), which then promotes translation initiation.
• control lifetime of RNA because while capped the mRNA is stable and protected from degradation. (decapping= degradation)

Question 7

List three reactions required to add a 5’ cap to pre-mRNA

Answer 7

The cap replaces the 5’ triphosphate on RNA. There are three enzymatic steps involved in adding the 5’ cap to m RNA:

1. A phosphatase (triphosphatase) removes the gamma phosphate from the 5’ end of the 5’ nucleotide.
2. Guanylyltransferase adds GTP to the terminal phosphate of RNA losing two phosphates from GTP in the process. This results in a 5’ to 5’ linkage between phosphates of RNA and GTP.
3. The 7 nitrogen of guanine ismethylated by a methyl transferase. S-adnosylmethionine is the methyl donor.

Question 8

What are the conserved sequences at the 5’ and 3’ ends of most introns and consensus sequence at the Poly A Site?

Answer 8

• 5’ end of intron consensus sequence: G U
• 3’ end of intron consensus sequence: A G
  (There is also a highly conserved branch point with an A residue.)
  Poly A Site: consensus sequence: AAUAAA approximately 20 bp downstream where the RNA is cleaved.

Question 9

Describe how alternative splicing permits multiple proteins to be produced by splicing defects.

Answer 9

Alternative splicing permits formation of multiple proteins from a single gene by utilizing different splicing which results in different mature mRNA transcript.
(There may be more codons, fewer codons, and frame shifts. Exons can be retained or excluded as in when one or more exons is skipped and excised.) Introns can also be retained in the sequence, creating additional C-terminus.

Question 10

Mutual Exclusion of an Exon

Answer 10

Inclusion of only one of two exons, but not both.

Question 11

Genetic Disorders Caused by Splicing Defects

Answer 11

Marfan Syndrome
Spinal Muscular Atrophy
Cancer (abnormal splicing of CD44 contributes to tumor metastasis, most BRCA1 defects are splicing related, contributing to breast cancer)

Question 12

Marfan Syndrome

Answer 12

Dominant autosomal disease caused by mutations that disrupt the splicing of the fibrilin gene transcripts. Many mutations are close to the 3’ or 5’ splice site, which interfere with binding of U1 and U2 snRNAs which regulate splicing. The result is mutant fibrilin, a component of connective tissues.

Signs and Symptoms:
Tall stature, long limbs, protruding sternum andweakness or defects of aorta and heart valves.

Question 13

**Describe the function of of U1 snRNAs in splicing.

Answer 13

U1 snRNA recognizes the 5’ splice sequence by base pairing with it along the transition from exon to intron.

Question 14

**Describe the function of of U2 snRNAs in splicing.

Answer 14

U2 snRNA recognizes the branch point by base pairing to it. (Branch point: a particular nucleotide sequence near the 3’ end of the intron that becomes covalently linked to the 5’ end of the intron during the splicing process)

Question 15

Identify Transcription Start Site, Introns, 5’ splice site and 3’ splice site, branch points on a diagram of a gene.

Answer 15

See diagram.

Question 16

What are the two reactions required to make the mature 3’ end of mRNAs?

Answer 16

1. Cleavage (RNA is cut by an endonuclease exposing a 3’ hydroxyl group)
2. Addition of Poly-A Tail (at 3’ OH a string of adenosines is added by polyadenyltae polymerase)

Question 17

Describe the relationship between the 3’ end processing of the pre-mRNA and termination of transcription at the end of the gene.

Answer 17

Formation of the 3’ end of mature mRNA by cleavage and polyadenylation is coupled with the termination of transcription by RNA polymerase II. Cleavage/ polyadenylation specificity factor (CPSF) is in contact with RNA pol II as it is transcribing. CPSF is also associated with polyadenylate polymerase. When the poly A tail is approximately 250 nucleotides long, the polyadenylate polymerase stops adding A’s and loses its affinity for CPSF and can no longer bind it. CPSF is no longer in contact with RNA pol II, thus signals to terminate txn.

Question 18

Describe the two major functions of the mRNA’s poly A tail.

Answer 18

1. mRNA protection/ stabiliazation: the poly A tail stabilizes the mRNA in the cytoplasm, preventing it from degraded prematurely.
2. Translatability of mRNA: the tail is bound by poly A binding protein I (PAB I) which boosts translation in the cytoplasm by helping recruit mRNA to ribosomes.
   Also helps export it from nucleus to the cytoplasm.

Question 19

Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.

Answer 19

A single gene can have more than one poly A site. Different locations of cleavage can be result in some exons being left out or truncated. Thus different exon arrangements cause different protein products in translation. In addition, alternative poly A sites can cause changes to the 3’ UTR, resulting in different micro RNA regulation. Usually, microRNA is a repressor of translation, therefore if 3’UTR is cleaved early, there is no repression, so there is overexpression of a particular gene. Common in various cancers.

Question 20

Immunoglobin M (IgM)

Answer 20

In the gene for the heavy chain of IgM, here are two poly A tail sites, a muS and an muM site. Cleavage and polyadenylation at the muS site results in an antibody form that is secreted from plasma cells. Cleavage and polyadenylation at the muM site results in a membrane bound antigen receptor on B cells.