Readiness Quiz #2 - RNA processing

Why is the 7mg cap important?

The 5’ end of the pre-mRNA is 7mg capped. 

Capping is important for binding other factors for export from the eukaryotic nucleus and for protein synthesis (translation) that will occur in the cytoplasm. 

The 7mg group has an unusual linkage 5’ to 5’. 

EXONS

coding sequences 

INTRONS

non coding sequences

How are intron sequences removed?

Through RNA splicing

Is splicing the same for a gene in all cells?

No. 

RNA can be spliced in many different ways to produce distinct mRNAs which will create different proteins. 

How does the cell know where to splice?

Specialized RNA molecules recognize the nucleotide sequences that specify where splicing is to occur and also catalyze the chemistry of splicing

What’s an examples of the same gene being spliced differently to have different functions?

a-Tropomyosin

 The α-tropomyosin made in striated muscle is different from that made from the same gene in smooth muscle. 

What is the spliceosome made of?

The spliceosome is made up of small nuclear RNAs (snRNAs). Each of them have seven protein subunits to form small nuclear ribonucleoprotein (snRNP). 

What are the sequences in the pre-mRNA that guide the spliceosome to correctly remove introns?

They are consensus nucleotide sequences that signal the beginning and the end of most introns. 

R = for purines (A or G) 

Y = for pyrimidines (C or U) 

A = the branch point of the lariat produced by the splicing 

GU highlights the start of the introns and AG highlights the end of the introns

The pre-mRNA splicing mechanism 

The spliceosome recognizes the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps required. 

What are the roles of the snRNA containing protein complexes? U1, U2, U4/6

U1 snRNP = forms base pairs with the 5’ splice junction and the (Branch point binding protein) BBP and U2AF (U2 auxiliary factor) recognize the branch point site. 

U2 snRNP = displaces BBP and U2AF and forms base pairs with the branch point site consensus sequence. 

U4/6*5 snRNP = snRNP “triple” enters the reaction - the U4 and U6 snRNAs are held firmly together by base pair interactions. Subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to display U1 at the 5’ splice junction. This creates the active site that catalyzes the first phosphoryl-transferase reaction. 

Exon Definition hypothesis

The SR proteins bind to each exon sequence in the pre-mRNA and thereby help to guide the snRNPs to proper intron/exon boundaries. 

SR proteins = rich in serines and arginine’s

Is the splicing of a pre-mRNA always 100% faithful- or do errors occur? 

It is not always 100% faithful.  The disease Beta Thalassemia which is a severe anemia is caused by splice site mutations. 

How is mature RNA exported from the nucleus?

Mature mRNA are guided through nuclear pore complexes to the outside of the cell. 

What is a nuclear pore?

Nuclear pores are aqueous channels in the nuclear membrane that directly connect the nucleoplasm and cytosol. 

Small molecules can diffuse freely through these channels. 

mRNA with attached proteins are too large to fit through without special processes. They are considered macromolecules and will need nuclear transport receptors. 

Do mature mRNAs just diffuse through the pore or is an export receptor needed?

For nuclear export to occur a specific nuclear transport receptor must be loaded onto the mRNA. This takes place at the same time as 3’ cleavage and polyadenylation. 

Once the transport receptor helps move an RNA molecule through the nuclear pore complex, the transport receptor dissociates from the mRNA, re-enters the nucleus, and is then used for a new mRNA transport. 

Where are non-coding RNAs synthesized? rRNA, snRNA, tRNAs

Non-coding RNAs (rRNAs, snRNAs, tRNAs) are synthesized and processed in the nucleus. 

Where does rRNA synthesis occur in the nucleus? 

In the nucleolus

The larger the S value..

The larger the rRNA

Which RNAs can be produced by the cleavage of 45S pre-rRNA?

18S, 5.8S, and 28S rRNAs

How is the 3’ poly A tail on pre-mRNA generated?

1. The polydenylation signal is recognized by the Cleavage and Polyadenylation Specificity Factor, looking for a specific sequence (AAUAAA) (recognition sequence) 

2. Cleavage and Polyadenylation Specificity Factor binds to this sequence. 

3. Cleavage factors I and II (CFI, CFII) cut the pre-mRNA downstream of AAUAAA. 

4. Poly A Polymerase (PAP) starts to add adenines to the new 3’ end. no template is needed 

5. Poly A binding proteins (PABPs) bind to the tail which speeds up the PAP activity.