Week 4: RNA Processing Flashcards
What are the different processing reactions involved in the maturation of mRNA?
Addition of the 5’ 7-methylguanosine cap, addition of the 3’ poly-A tail, cleavage of the tail, splicing, and alternative assembly of spliced elements
Compare and contrast self-splicing and spliceosome-dependent splicing
Group I and II introns are self-splicing, and follow the “UAGU, AG” splicing connection mechanism.
Group III introns are spliced by spliceosomes, and are the most common introns. They follow the “GU(L)AG” splicing series. They also require ATP
What are the essential features of a spliceosome-dependent intron. What will happen if it is modified?
There is a GU(Link)AG sequence that U1 (GU) and U2 (AG) can bind to.
U2 must also bind to an Adenosine to help it bulge out in the mismatched RNA lineup
3 ATP are required to complete splicing
What is meant by alternative splicing? What are some examples?
Alternative splicing occurs as single genes yield different peptides depending on RNA processing
This occurs as regions may be retained or removed, yielding different mature transcripts
Examples include calcitonin in the thyroid and calcitonin-gene-related peptide (CGRP) in the brain
What are some mechanisms of mRNA quality control and degradation? What are some examples?
No-go mRNA decay occurs when ribosomal translation is blocked by a strong RNA structure that stalls Pol II, signaling for degradation
Nonsense-mediated mRNA decay occurs when Pol II encounters a premature stop codon (PTC) without also contacting an exon junction complex. If Pol II contacts a PTC without an EJC, the protein is targeted for degradation
Nonstop mRNA decay occurs when Pol II runs into the poly-A tail, and triggers degradation
What are the three major steps in the processing of eukaryotic mRNA?
Adding the 5’ cap, addtion of the noncoding, poly-A tail, splicing of introns, and alternative assembly of introns
What are the protective elements at the 5’ end of mRNA?
The 7-methylguanosine cap at the 5’ end, formed with a molecule of GTP
Methylations at the 2’ -OH groups of the next two nucleotides after the cap
How does the 5’ end secure itself to Pol II?
The cap-synthesizing complex binds to the phosphorylated CTD, and then binds the 5’ end of mRNA.
It is then swapped for the cap-binding complex (CBC)
What are the steps of Group I intron splicing?
(1) The 3’-OH of a free GMP/GDP is used as a nucleotide to split a UA sequence in half, with Adenosine nucleotide breaking off and binding to guanosine
(2) the -OH bound to uracil on the end of exon 1 nucleophilically attacks the GU sequence on the start of exon 2, completing the reaction
(3) The guanosine in the GU sequence splits off with the intron, leaving the two uracil moieties bound to one another as exons 1 and 2
What are the steps in Group II intron splicing?
(1) The 2’ OH of an adenosine in the intron nucleophilically attacks a UG sequence at the end of exon 1, forming a lariat structure
(2) The -OH bound to the uracil now at the end of exon 1 nucleophilically attacks the uracil at the start of exon 2
(3) The reaction is complete, and the intron breaks away in the lariat structure
Explain the steps of Group III splicing
(1) Using one ATP for energy, the U1 and 2 snRNPs bind to the GU/A/AG sequences at either end of the intron
(2) Using another ATP, U4, 5, and 6 ind to the U1/2 complex, foring an inactive spliceosome
(3) The mismatched RNA lineup creates a bulge at an adenosine nucleotide
(4) The A at the far end of the intron is brought into contact with the GU nucleotides at the beginning, nucleophilically attacking and breaking them away from the end of exon 1. At the same time, an ATP is expended to expel the U1 and U4 sNRPs, which helps bring the GU and A together.
(5) U5, which secures the A/AG moieties at theend of the intronbreaks away with the lariat structure, with theGUfrom thebeginning of theintron, leaving the -OH at the end ofexon 1to nucleophilically attack the3’ end of exon 2 and conjoin
How does the spliceosome interact with RNA Pol II?
Some components of the spliceosome are attached to the CTD of RNA Pol II. This coordinates splicing and transcription simultaneously, and allows for the sequential, ordered binding of introns to one another (i.e. 1 to 2 to 3, instead of 3 to 11 to 6 or some random order)
How does RNA Pol II add the poly-A tail?
The polyadenylation factors at the CTD of Pol II add A repeats, and also synthesizes the tail beyond the AAUAAA cleavage sequence
The cleavage signal is bound by an endonuclease and a polyadenylate polymerase on the CTD
The endonuclease cleaves RNA 10-30 nt downstream of the AAUAAA sequence
Polyadenylate polymerase adds 80-250 adenosine nts to protecc mRNA from degradation and to increase the efficiency of translation
What is the 5’ cap and how is it bound? What is it’s main role as it relates to moving the mRNA from the nucleus to the cytoplasm?
The 5’ cap is a 7-methylguanosine molecule bound to 3 phosphates to the 5’ end of the developing RNA transcript. It is formed by the attachment of a GTP, making a 5’-5’ triphosphate linkage. This is catalyzed by guanylyl transferase, which is closely associated with the C-terminal domain of RNA polII so that the enzyme can act almost immediately once the RNA transcript starts forming. This is all completed by the cap-synthesizing complex.
What occurs after the 5’ cap has been formed?
Once the cap has formed, it dissociates, and the cap binding complex (CBC) attaches to the CTD, securing the 5’ cap as transcription occurs.
The 5’ cap is critical for protecting the primary transcript and delivering it to the ribosome for translation.
Explain the process of alternative splicing
A single gene can yield different peptides depending on how the primary RNA transcript is processed. Splicing uses the same foundational DNA, but omits certain segments based on the tissue type, developmental or growth conditions, and what proteins are needed. Two major kinds are alternative 5’ or 3’ splice site selection (left) or a change in the polyA site, which can lead to a polyA tail on a shorter RNA sequence or a longer one (right).
