RNA Processing and Post-Transcriptional Gene Regulation

Question 1

RNA processing refers to any — to the RNA after transcription

Answer 1

covalent modification

Question 2

Steps of RNA processing?

Answer 2

1. RNA cleavage at a specific site
2. 5’ CAP
3. splicing (intron removal)
4. poly(A) tail

Question 3

5’ capping, 3’ polyA addition and splicing are all

Answer 3

coupled to transcriptional machinery (as RNA is being made)

Question 4

How does 5’ capping occur?

Answer 4

1. starts 20-30 nt after transcription initiation
2. remove gamma phosphate of the first nucleotide
3. link a GMP to the beta phosphate from a GTP using guanylyltransferase
4. N-7 of GMP is methylated using methyltransferase (CH3 from S-adenosylmethionine)
5. methylation of 2’OH of first 1-2 nucleotides of mRNA

structure of 5’ cap = 7’methyl-guanosine

Question 5

Known interactors of CTD:

Answer 5

• enzymes involved in processing of 5’ & 3’ ends
• splicing factors
• chromatin remodeling factors
• export proteins

CTD of RNAPII needs to be phosphorylated

Question 6

The 5′ cap has four main functions:

Answer 6

1. Regulate nuclear export
2. Prevent degradation by exonucleases
3. Promote 5′ proximal intron excision
4. Interact with ribosome and promote of translation

Question 7

How does polyadenylation factors know when to bind

Answer 7

3’ end usually AT rich which is recognized by polyadenylation factors

Question 8

How do we identify the 5’ end (TSS) of an mRNA?

Answer 8

S1 nuclease mapping
- hybridize target mRNA to 5’-labeled DNA
- DNA-RNA duplex with ssRNA and ssDNA on each end
- S1 nuclease cuts off all ssDNA and ssRNA
- run on gel to determine size
- compare with and without S1 nuclease bands

Question 9

How can you identify 5’ end of mRNA that is low abundance?

Answer 9

primer extension:
- mRNA transcript hybridized to a labeled primer
- extend primer with reverse transcriptase
- electrophorese to find start site
- use RACE PCR

Question 10

Polyadenylation process?

Answer 10

1. mRNA transcripts have AU rich region newly transcribed
2. that is bound by polyadenylation factors
3. proteins cleave RNA
4. PAP makes poly A tail
5. PABP protects tail

Question 11

Purpose of polyA tail?

Answer 11

• adds stability, increasing the time during which the mRNA (remains intact and available for translation before cellular enzymes degrade it)
• enhances the ribosome’s attachment to the mRNA

Question 12

What are the different kinds of introns?

Answer 12

1. Group I introns
2. Group II introns
3. Spliceosomal introns

Question 13

What is a Group I introns?

Answer 13

found in bacteria, mitochondria, chloroplasts, and some nuclear rRNA genes - self-splicing, initiated by a guanosine cofactor
- circle lariat
- GTP becomes covalently attached to the spliced intron

RNA catalyst

Question 14

What is a Group II intron?

Answer 14

found in bacteria, mitochondria, chloroplasts - self-splicing, initiated by an internal adenosine
same as spliceosome mech, without spliceosome

RNA catalyst

Question 15

What are spliceosomal introns?

Answer 15

found in eukaryotic nuclear protein-coding genes - splicing requires a spliceosome

Question 16

boundaries between introns and exons are called an

Answer 16

intron-exon junctions

Question 17

Majority of introns in nuclear protein-coding genes follow the – rule

Answer 17

5’ splice site GT-AG 3’ splice site

(or GU-AG if referring to RNA)

Question 18

What is the reaction that occurs for intron splicing?

Answer 18

2 transesterification reactions
- OH of A branch point attacks phosphate at 5’ splice site making lariat structure
- OH of 3’ of exon 1 attacks phosphate at 3’ splice site

Question 19

protein-RNA complex that mediates splicing in eukaryotes

Answer 19

spliceosome

Question 20

composition of spliceosome?

Answer 20

• proteins, pre-mRNA, 5 snRNA
• snRNAs form ribonucleoparticles (RNPs) with spliceosome-associated proteins = snRNPs

Question 21

Base-pairing between the — mediates recognition of splice boundaries

Answer 21

individual snRNAs and exon-intron sequences

Question 22

alternative splicing

Answer 22

Genes with multiple exons may give
rise to transcripts containing different
combinations of exons

~25,000 genes, millions of protein

Question 23

5 patterns of alternative splicing?

Answer 23

1. alternative 5’SS
2. alternative 3’SS
3. alternative exon skipping
4. alternative intron retention
5. mutually exclusive exons