RNA Processing Flashcards
How many RNA polymerases are there in eukaryotes and what do they transcribe?
3
Pol I - rRNA (main)
Pol II - mRNA (main), snRNA, miRNA
Pol III - tRNA (main), 5S rRNA, other small RNAs
What are the different types of RNA processing?
Cleavage, Splicing, 5’ capping, polyadenlyation, base insertion, deletion, modification
What are 2 characteristics of rRNA genes in the genome?
Polycistronic messages and found repeated in all genomes.
How are rRNAs primarily processed?
Through endo and exonucleolitic activities.
List the 5 ways tRNAs are processed post-transcriptionally in eukaryotes?
- 5’ cleavage by RNase P
- 3’ cleavage by tRNase Z
- CCA is added to 3’ end by enzyme
- Post-transcriptional modifications of bases
- Intron excision
What are two common post-transcriptional modifications of rRNA in eukaryotes and which snoRNAs mediate those modifications?
- 2’ O methylation by box C/D snoRNA
2. Pseudouridylation by H/ACA snoRNA
How do snoRNAs facilitate modification of nucleotides?
In a template driven manner. snoRNAs bind to complementary sequence and position nucleotide for modification within active site.
What is odd about the 5’ cap found on mRNAs?
The guanosine is attached to the first base in a 5’ -> 5’ linkage
What are the 3 steps involved in 5’ capping?
1) Removal of terminal phosphate from nascent RNA
2) Addition of GMP to the end, forming a 5’ -> 5’ linkage
3) Methylation of guanine
What binds to the poly A tail?
Poly A Binding Protein (PABP)
What can be used to enrich mRNA transcripts from lysates and why is this significant?
Use Poly-A tail by pulling down with Poly-T oligos. only 3% of total cellular RNA is mRNA. Majority is rRNA
Which mRNAs do not have poly A tails and how do they get around this?
Histone mRNAs. Have a 3’ end stem loop generated by U7 snRNP-mediated interaction. Stem loop is bound by its own protein instead of PABP
5 roles for 5’ cap and poly A tail
- Help downstream maturation including splicing and transcription termination
- mRNA stability
- Facilitate transport to cytoplasm
- Important for translation initiation
- Alternative polyA/capping can be regulatory
What consensus sequences are recognized by the splicing machinery?
5’ splice site GU
3’ splice site AG
Branch point A
Purine Rich Sequence downstream branch point
Describe or draw the 3 steps of pre-mRNA splicing?
1) 2’ OH of branch point Adenosine attacks 5’ splice site
2) Free 3’ OH of exon attacks 3’ splice site.
3) Get lariat (2’5’) intermediate that’s degraded
Note: not energetically demanding
What is the main part of snRNPs that catalyze reactions? What is the theme associated with the spliceosome?
RNAs. The spliceosome isn’t a large machine that works all at once. It consists of smaller RNPs that dynamically lead to splicing.
How do the mechanisms of Group I and Group II introns differ?
Group I introns use an exogenous guanosine to initiate attack 5’ splice site. Group II introns use branch point adenosine, similar to how introns are spliced in eukaryotes.
What happens if you mutate the 5’ splice site of an intron in higher eukaryotes?
You get exon exclusion. Exons are defned in higher eukaryotes.
What happens if you mutate the 5’ splice site of an intron in lower eukaryotes?
You get intron inclusion. Introns are “defined” in lower eukaryotes.
How are the strengths of splice sites modulated in different tissues?
Differential expression of RNA binding proteins (such as intronic silencers) can change the splice site that will be used. This can cause proteins to be functional or non-functional trough alternative splicing.
What is an example of RNA editing?
Nucleotide addition and deamination
How is RNA canonically destroyed in eukaryotes?
PAPB disassociates and a 3’-5’ exonuclease / exosome cleaves one end.
Another enzyme decaps the mRNA and Xrn1 cleaves 5’->3’ with exonuclease.
