Lecture 9: RNA Structure and Processing (Post-Transcription)

Question 1

Is RNA more malleable than DNA

Answer 1

Yes

Question 2

RNA is mostly ___ stranded but it can ____ with ______ within itself

Answer 2

1. Single
2. Base-pair
3. Complementary Sequences

Question 3

This is know as the code for the synthesis of proteins and a template for translation

Answer 3

mRNAs

Question 4

What is rRNA and what is its role/function?

Answer 4

• rRNA is the basic structure of the ribosome, it is embedded in the protein
• catalyze protein synthesis
• Ribosome = rRNA and other proteins
• rRNA = ribosomal RNA

Question 5

What is tRNA and what is its role/function?

Answer 5

• Acts as an adapter between mRNA and amino acids
• Has a hairpin structure/format
• Important in protein synthesis
• tRNA = transfer RNA

Question 6

What is snRNA and what is its role/function?

Answer 6

• Involved in pre-mRNA splicing (attach to proteins to form snRNPs/spliceosome)
• Also forms hairpins and loops with itself – base-complements with itself
• snRNA = small nuclear RNA

Question 7

What is snoRNA and what is its role/function?

Answer 7

• Processing and chemical modification of pre-rRNAs
• In the nucleolus
• snoRNA = small nucleolar RNA

Question 8

What is miRNA and what is its role/function?

Answer 8

• Regulate gene expression by blocking translation of specific mRNAs and targets it for degradation
• miRNA = micro RNA
• base-pair extensively, but not completely, with mRNAs

Question 9

What is siRNA and what is its role/function?

Answer 9

• Together with associated proteins, siRNAs cause cleavage of the target RNA, leading to its rapid degradation
• siRNA = short interfering RNA
• each perfectly complementary to a sequence in an
  mRNA

Question 10

What is piRNA and what is its role/function?

Answer 10

• Bind to piwi proteins and protect the germ line from transposable elements.
• Piwi proteins – important for protecting germ lines
• piRNA = Piwi-interacting RNAs

Question 11

What is lncRNA and what is its role/function?

Answer 11

• Scaffolds for chromatin folding; X-chromosome inactivation, and much more!
• lncRNA = long non-coding RNA

Question 12

What is the difference between mRNA and pre-mRNA?

Answer 12

pre-mRNA: an mRNA precursor containing introns and not cleaved at the poly(A) site
mRNA: Fully processed messenger RNA with 5ʹ cap, introns removed by RNA splicing, and a poly(A) tail.

Question 13

What is pre-tRNA?

Answer 13

A tRNA precursor containing additional transcribed bases at the 5ʹ and 3ʹ ends compared with the mature tRNA. Some pre-tRNAs also contain an intron in the anticodon loop.

Question 14

What is pre-rRNA?

Answer 14

The precursor to mature 18S, 5.8S, and 28S rihosomal RNAs. The mature rRNAs are processed from this long precursor RNA molecule by cleavage, removal of bases from the ends of the cleaved products, and modification of specific bases.

Question 15

How is RNA (RNA from protein coding genes) processed?

Answer 15

Before first step:
- During transcription, post-transcriptional processes also occur e.g. 5’ cap added 20-30 nucleotides into the transcript
- Transcribed from RNA Pol II
Process:
1. Pre-mRNA splicing: use of alternative exons during pre-mRNA splicing
2. Cleavage/polyadenylation: Use of alternative poly(A) sites. The poly A tail prevents exonuclease from degrading pre-mRNA
3. Correctly processed mRNA VS Improperly processed
- Properly processed mRNAs – exported to the cytoplasm
- Improperly processed mRNAs – blocked from export to the cytoplasm and degraded the exosome complex containing multiple ribonucleases
4. mRNA export and translation initiation factors: translation initiation factors bind to the 5ʹ cap cooperatively with poly(A)- binding protein I bound to the poly(A) tail and initiate translation

Question 16

What does RNP stand for and why are they important

Answer 16

• RNP = ribonucleoproteins
• RNPs are important in catalyzing splicing

Question 17

How are two exons joined together in splicing?

Answer 17

A large ribonucleoprotein spliceosome complex catalyzes two transesterification reactions that join two exons and remove the intron as a lariat structure

Question 18

How do they know where to splice on the pre-mRNA

Answer 18

A network of interactions between SR (serine-arginine rich) proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that specifies correct splice sites

Question 19

Summarize what happens to pre-mRNA before it is exported to the cytoplasm

Answer 19

Pre-mRNA is capped, polyadenylated, spliced, and associated with RNPs (ribonucleoproteins) in the nucleus before export to the cytoplasm.

Question 20

For the G-value paradox, explain one of the reasons why gene number does not correlate with complexity

Answer 20

Due to differential processing of pre-mRNAs! – this process increases amount of diversity. Example: alternative splicing allows one gene to give rise to a mRNA transcript that can be spliced to give rise to multiple different mRNA (aka different proteins)

Question 21

What is constitutive splicing

Answer 21

removes all introns, fuses all exons

Question 22

What is exon skipping in splicing

Answer 22

not all exons included

Question 23

What does it mean to be mutually exclusive in splicing

Answer 23

two exons are never together in a given transcript

Question 24

What does an alternative 5’ and alternative 3’ mean in splicing

Answer 24

different splice factors determine where the mRNA starts and/or ends so different areas can be spliced out

Question 25

What is the largest human gene

Answer 25

Contactin-associated protein-like 2 gene (CNTNAP2)

Question 26

Human gene with the most exons

Answer 26

TTN (titan) gene, an essential component of the sarcomere

Question 27

What is the goal of RNA processing in eukaryotes

Answer 27

Producing a functional mRNA

Question 28

How does eukaryotic mRNA difference from prokaryote due to RNA processing

Answer 28

Prokaryote mRNA has no poly A tail and its 5' cap is not methylated

Question 29

What is an UTR in mRNA

Answer 29

- It is an untranslated region inn the fully processed mRNA - It can be found on the 5' and 3' end but during translation it is upstream/downstream of start/stop region so it is not translated

Question 30

How is the poly A tail formed? What is the function the poly A tail?

Answer 30

- Primary transcript is cleaved at a specific site downstream of the translation STOP codon, and multiple (100–200) A residues (not encoded by the gene DNA template) are added enzymatically by poly(A) polymerase - Poly(A) tail stabilizes mRNAs in the nucleus and cytoplasm and is involved in mRNA translation

Question 31

What is the function of the 5' cap? What is the name of the methylated cap?

Answer 31

- Protects mRNA from enzymatic degradation (specifically from exonuclease activity), assists export to the cytoplasm, and is bound by a protein factor required to begin translation by a ribosome in the cytoplasm. - Name = 7-methylguanylate

Question 32

How is the 5' cap synthesized?

Answer 32

- Protective 5’ cap added as a nascent eukaryotic RNA transcript emerges from the Pol II RNA exit channel and reaches a length of about 25 nucleotides Step 1: Initial modification: - γ phosphate – removed from the first base by dimeric capping enzyme (phosphohydrolase activity) associated with the RNA polymerase II phosphorylated CTD α and β phosphates – remain associated with the cap 5’ cap interacts with RNA polymerase II Step 2: Guanylyl transferase links GMP from GTP to the 5ʹ diphosphate of the nascent transcript – creates a guanosine 5ʹ-5ʹ triphosphate structure Three phosphates = 5’ to 5’ linkage in image in previous slides Step 3: Guanylyl-7-methyl transferase transfers methyl group from S- adenosylmethionine to the guanine N7 position Step 4: 2’-O-methyl transferases transfers methyl group from S- adenosylmethionine to the 2ʹ oxygens of riboses of the first one or two RNA nucleotides