Discussion Quiz 9 RNA Processing

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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


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