Discussion Quiz 9 RNA Processing
How many common pathways for degradation of mRNA are present in eukaryotes? Are they sequence specific? What does it mean if they are not sequence specific?
2; No, it can happen to any mRNA; The same mRNA can be degraded in either pathway
Assuming no mutations, how many different RNA species could theoretically be produced from a transcription unit that contains 3 exons and 2 introns?
4
Which would you expect to have a longer half-life, a prokaryotic or eukaryotic mRNA, and why?
A eukaryotic mRNA - has more stabilizing structures
Does the transcript end after, at, or before the AAUAAA site?
After
Description of microRNA-mediated silencing
Binding of complementary microRNA with RISC complex via the RISC endonuclease (5'-3', 3'-5')
PABP (Poly(A) binding protein)
Binds poly(A) tail and confers message stability, helps with translation
What is the function of cleavage factors (CFI and CFII)?
Both are endonucleases that cleave RNA
Nucleases:
DNase, RNase, endonucleases, exonucleases (5'-3' and 3'-5')
What is the name of the multicomplex structure in bacteria that is responsible for RNA degradation and what is the analogous structure in eukaryotes?
Degradosome - bacteria Exosome - eukaryote
Eukaryotes: mRNA relative stability - mRNA lifespan - Stabilizing structures - Message is degraded when... Location of transcription and translation - General degradation mechanisms -
Eukaryotes: mRNA relative stability - higher (up to 24 hours) mRNA lifespan - minutes to days Stabilizing structures - 5' cap (guanine) and poly-A tail Message is degraded when... the poly A-tail is shortened to 10 adenines Location of transcription and translation - transcription is in the nucleus and translation is in the cytoplasm General degradation mechanisms - deadenylation-dependent capping (5'-3' decay), exosome-dependent (3'-5' decay)
What are the important sequences in snRNAs?
GU (donor site), AG (acceptor site), A(Branch site)
Which elements in introns are conserved?
GU of donor, AG of acceptor, Branch (one of the adenines)
What is the function of cleavage stimulatory factor? (CstF)
It binds CPSF and recruits endonucleases CFI and CFII
What is the function of poly(A) polymerase, aka PAP?
It binds the 3'end and adds a long chain of adenines
Why is RNA less stable than DNA?
It is single-stranded and they are degraded more frequently.
What is the function of cleavage and polyadenylation specificity factor (CPSF)?
It recognizes the AAUAAA sequence and recruits CstF
Do all eukaryotic messages have 5' to 3' UTRs?
No
Do all genes in eukaryotes have introns?
No
Do prokaryotes have introns? Do prokaryotes use the spliceosome assembly?
No and no
Are introns removed in order? Are exons spliced in order?
No, it's random. Yes, exons ARE spliced in order. If not, the gene may change or become nonfunctional
What is the general function of microRNAs?
RNA silencing
What is RNAi?
RNA-dependent gene regulation (suppression) in living cells; used to silence genes.
What molecular catalyzes nucleophilic attacks?
RNP (ribonucleoprotein)
What's so unusual about the 5' cap found on eukaryotic mRNA? What's special about its linkage?
The 5' cap is a methylated guanine with a 5'-5' linkage. It retains the triphosphate. The linkage is 5' to 5'.
Every time a nucleotide is added to a growing RNA, what part of the new nucleotide is lost? What is an exception to this?
The PPi of the new nucleotide is lost. An exception is the 5' cap.
Among different snRNP "snurps", do they contain similar or different proteins?
They contain both - 8 common (conserved) proteins and 12 unique proteins (different between organisms)
What is the role of the AAUAAA sequence once it becomes transcribed into the RNA chain?
This sequence initiates the formation of the poly-A tail.
What is the purpose of the splicing complex?
To recognize introns, remove the introns, and splice the exons
Destabilizing sequence: Type of molecule - Usual location - General Function - Specific functions -
Type of molecule - mRNA Usual location - 3' UTR General Function - decreases half-life of mRNA Specific functions - recruit RNA binding proteins that interact with decapping enzymes, deadenylation enzymes, and/or exosome
Stabilizing sequence: Type of molecule - Usual location - General Function - Specific functions -
Type of molecule - mRNA Usual location - 3' UTR General Function - increase half-life of mRNA Specific functions - recruit stabilizing proteins like PABP
Why is turning genes off by RNAi easier than creating mutations?
We don't need to alter the DNA
Can degradation occur without removal of the poly-A tail?
Yes via deadenylation-independent decapping
Can some introns have AU instead of a GU sequence at the donor site?Would that site be recognized by the "usual" splicing factors?
Yes, it can; No, it wouldn't be recognizd by the usual splicing factors.
Does splicing require a particular shape OR a particular sequence?
Yes, it requires a particular sequence
Function of endonuclease
cleaves nucleic acids at internal sites
What factors that control gene expression are microRNAs affecting?
degradation of mRNA, inhibition of translation
Function of DNase
degrades DNA
Function of RNase
degrades RNA
What is a sequence that decreases the stability of mRNA called? What type of molecule is it?
destabilizing sequence; mRNA
What type of nuclease makes a cut after the AAUAAA site?
endonucleases CFI and CFII --> both cut at the cleavage sites
where are microRNAs found in the genome?
everywhere
What happens if a miRNA is full complementary/partially complementary to the target mRNA?
fully complementary --> degradation of mRNA partially complementary --> translation is inhibited
What enzyme adds the cap?
guanylyl tranferase
Prokaryotes: mRNA relative stability - mRNA lifespan - Stabilizing structures - Message is degraded when... Location of transcription and translation - General degradation mechanisms -
mRNA relative stability - lower (1-3 min) mRNA lifespan - minutes Stabilizing structures - stem loop Message is degraded when... ribsosomes are NOT densely packed Location of transcription and translation - same place (cytoplasm) General degradation mechanisms - mRNA is degraded by degradosome
What are other modification at the 5' end besides the addition of the G nucleotide that contributes to the cap structure?
methylation of guanine
What is the general half-life of a bacterial RNA? What can extend or decrease the lifespan of the message?
minutes; ribosomes that are densely packed + stem loops
What might long (+26 bps) dsRNA trigger in eukaryotes?
nonspecific degradation
What is the structure called when many ribosome are translating one message?
polysome
Short temporal RNA (mi/stRNA)
produced developmentally to block expression of target genes
What is an RNP?
ribonucleoprotein --> snRNA + splicing factors
What are microRNAs?
small noncoding RNAs, complementary to target RNAs
What is the difference between snRNA and snRNP?
snRNA has no protein association, while snRNP does have protein association.
What are the components of the spliceosome?
splicing factors (proteins) and snRNPs 8 common structural proteins, 12 unique proteins
What is a spliceosome?
splicing factors + snRNPs recognition+removal of introns and ligation of exons Stages of the spliceosome happen in the following order: EABC
Piwi-associated RNA (piRNA) -
suppresses the action of transposable elements
Small interfering RNA (siRNA) -
suppresses viral and transposable element infection
