RNA Maturation

Question 1

Steps in RNA maturation

Answer 1

1. 5’ capping
2. Splicing and/or editing
3. 3’ polyadenylation
4. Transport out of the nucleus (becomes mature mRNA at this point)

Question 2

Hybridizing mature mRNA to DNA

Answer 2

Huge loops are present in the DNA: introns

Question 3

Size of introns

Answer 3

Vary in size: 100 bp- 800 kbp

Average is about 3500 bp

Question 4

Average size of exon

Answer 4

150 bp

Question 5

Splicing occurs…

Answer 5

Co-transcriptionally

Question 6

Complexity of an organism and intron numbers

Answer 6

More complex organism, more introns

Question 7

Determining which part of a gene is an intron versus an exon

Answer 7

Bioinformatics (looking for well-conserved sequences that control splicing)
Compare sequence of cDNA to gDNA
Compare RNAseq (transcriptome) to next gen sequencing of genome (coding regions of DNA)

Question 8

Roles that an intron plays

Answer 8

Some contain regulatory elements
Some code for siRNA
Allow for alternative splicing
Buffer against mutation

Question 9

Splice site consensus sequences

Answer 9

5’ and 3’ end: G’s
Branch site (approximately middle of intron): Adenine
If mutated, splicing doesn’t work
Play an active role in splicing (other sequences are merely recognized)

Question 10

2 step model of splicing

Answer 10

1. 2’ -OH of branch site attacks -PO3 of 5’ end of intron, forming a loop
2. -OH of 3’ end of 1st exon attacks -PO3 of 5’ end of 2nd exon, creating the intron lariat (degraded) and linking the two exons together

Question 11

Spliceosome components

Answer 11

snRNPs (small nuclear riboproteins, which are made up of snRNA and proteins)
Splicing factors

Question 12

How spliceosome locates intron

Answer 12

Complementary base pairing between snRNA and intron

Question 13

Where snRNPs are assembled

Answer 13

Cajal bodies (area of the nucleus)
snRNA meets up with its protein partner

Question 14

Steps in splicing by the spliceosome

Answer 14

1. U1 and U2 snRNPs bind using consensus sequences
2. Tri snRNP particle binds
3. RNA folds into precise configuration
4. 1st phosphodiester bond is broken
5. U4 snRNP dissociates via action of DEAD helicase
6. 2nd phosphodiester bond is broken
7. Intron lariat is degraded

Question 15

Percentage of human genome that is alternatively spliced

Answer 15

~60-90%

Question 16

Advantages that alternative splicing provides for an organism

Answer 16

Specific environmental responses

Less DNA to maintain

Question 17

Exonic splicing enhancer sequences (ESEs)

Answer 17

Splicing activators that promote the use of a particular splice site bind to these

Question 18

Exonic splicing silencer sequences (ESSs)

Answer 18

Splicing repressors that reduce the usage of a particular site bind to these

Question 19

Steric hindrance

Answer 19

Splice site selection mechanism
When an intron is small, only half of the spliceosome can bind to it, meaning that the exon bordering the intron that isn’t completely covered is excised along with the intron

Question 20

Major and minor splice sites

Answer 20

Splice site selection mechanism
Minor and major splice sites differ in consensus sequences: major spliceosome can’t bind to minor splice sites, or vice versa
One intron might have both major and minor splice sites

Question 21

Nonsense mediated decay

Answer 21

Defense against improper splicing
If the exons are spliced such that the mature mRNA contains a premature stop codon, then the RNA is targeted for destruction

Question 22

Docking: selector basepairing

Answer 22

Splice site selection mechanism
Occurs in Down Syndrome Cell Adhesion Molecule (DSCAM) gene
Docking site and selector sequence base pair, bringing exons that weren’t previously near each other into close proximity

Question 23

Self-splicing group I introns

Answer 23

Free G nucleotide is bound by RNA, enabling cleavage of the intron

Question 24

Self-splicing group II introns

Answer 24

Operates in a similar mechanism to the spliceosome

RNA folds into a structure that allows for splicing