RNA Maturation Flashcards
Steps in RNA maturation
- 5’ capping
- Splicing and/or editing
- 3’ polyadenylation
- Transport out of the nucleus (becomes mature mRNA at this point)
Hybridizing mature mRNA to DNA
Huge loops are present in the DNA: introns
Size of introns
Vary in size: 100 bp- 800 kbp
Average is about 3500 bp
Average size of exon
150 bp
Splicing occurs…
Co-transcriptionally
Complexity of an organism and intron numbers
More complex organism, more introns
Determining which part of a gene is an intron versus an exon
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)
Roles that an intron plays
Some contain regulatory elements
Some code for siRNA
Allow for alternative splicing
Buffer against mutation
Splice site consensus sequences
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)
2 step model of splicing
- 2’ -OH of branch site attacks -PO3 of 5’ end of intron, forming a loop
- -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
Spliceosome components
snRNPs (small nuclear riboproteins, which are made up of snRNA and proteins)
Splicing factors
How spliceosome locates intron
Complementary base pairing between snRNA and intron
Where snRNPs are assembled
Cajal bodies (area of the nucleus) snRNA meets up with its protein partner
Steps in splicing by the spliceosome
- U1 and U2 snRNPs bind using consensus sequences
- Tri snRNP particle binds
- RNA folds into precise configuration
- 1st phosphodiester bond is broken
- U4 snRNP dissociates via action of DEAD helicase
- 2nd phosphodiester bond is broken
- Intron lariat is degraded
Percentage of human genome that is alternatively spliced
~60-90%
Advantages that alternative splicing provides for an organism
Specific environmental responses
Less DNA to maintain
Exonic splicing enhancer sequences (ESEs)
Splicing activators that promote the use of a particular splice site bind to these
Exonic splicing silencer sequences (ESSs)
Splicing repressors that reduce the usage of a particular site bind to these
Steric hindrance
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
Major and minor splice sites
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
Nonsense mediated decay
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
Docking: selector basepairing
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
Self-splicing group I introns
Free G nucleotide is bound by RNA, enabling cleavage of the intron
Self-splicing group II introns
Operates in a similar mechanism to the spliceosome
RNA folds into a structure that allows for splicing
