L10: RNA Splicing Flashcards
define exon
- gene sequence retained in a mature RNA
- mostly coding sequence
define intron
- gene sequence absent from mature RNA
- non-coding sequence
pre-mRNA vs spliced mRNA
- pre-mRNA: contains introns
- spliced mRNA: introns removed
- none of these are mature RNA
define RNA splicing
- process of intron removal
- only happens in eukaryotes since they are the only ones that have introns
RNA splicing - what are other processing?
addition of 5’ cap and 3’ polyA tail
what are the important sites for chemistry of RNA splicing?
- 5’ splice site
- 3’ splice site
- branch site
chemistry of RNA splicing - 5’ splicing site
- makes exon-intron boundary
- 5’ end of intron is typically GU
chemistry of RNA splicing - 3’ splice site
- marks the intron-exon boundary
- 3’ end of intron is typically AG
chemistry of RNA splicing - branch site
single nucleotide (typically A) in the intron followed by a polypyrimidine tract (Py tract)
chemistry of RNA splicing - intron removal
- happens on branch site
- there are two transesterification reactions during intron splicing (transfer of phosphodiester bonds need to change)
- unique bc it makes 3 linkages not just 2
- 5’ end of of intron interacts with A and get three points where A is linked to RNA
what proteins are mediating intron removal
- spliceosome
- snRNAs
- snRNPs
proteins mediating - spliceosome
- huge molecular machine that mediates transesterification reactions
- made of 150 proteins and 5 RNAs
proteins mediating - snRNAs
- small nuclear RNAs
- U1-6
- complexes with proteins to form small nuclear ribonuclear proteins (snRNPs)
proteins mediating - snRNPs
- recognize splice and branch sites
- brings ends together
- catalyze RNA cleavage and joining
what is alternative splicing?
- deliberately skipping exons or retaining introns
- can have an exon skipped, extended, an
- creates genetic diversity and can produce different isoforms in certain tissues or at varying times
- over 90% of human mRNAs undergo alternative splicing
alternative splicing - what can the spliced mRNA look like?
- exon skipped
- exon extended
- intron retained
- alternative exons
example of alternative splicing - extended exon of T antigen from monkey virus SV40
- one gene encodes both large (T-ag) and small (t-ag) T-antigens
- complete removal of intron generates full length T-ag product
- retaining intron portion to give an extended exon generates t-ag
protein regulators of alternative splicing
- SR proteins
- hnRNP
protein regulators of alternative splicing - SR proteins
- activators
- has two domains:
1. RNA-recognition motif that binds splicing enhancers
2. domain rich in arginine and serine (RS domain) that recruits components of spliceosome
what is the exon splicing enhancer (ESE)?
- sequences in the exon that promote splicing by recruiting SR proteins
- Mutations in ESEs can lead to splicing modulation (exon skipping or inclusion), which is associated with various genetic diseases
- skipping is more common
protein regulators of alternative splicing - hnRNP
- heterogeneous nuclear ribonuclear protein
- repressor that binds to RNA but the RNA lacks RS domains
- therefore blocks splicing sites
Drosophila sex determination by alternative splicing
- XY = males; XX = females
- X chromosome encodes a transcription factors that activate an autosomal gene Sex-lethal (Sxl)
Drosophila sex determination by alternative splicing - Sxl
- a splicing repressor
- its a critical gene (master regulator) in sex determination
Drosophila sex determination by alternative splicing - what transcription factors are used?
- SisA and SisB: encoded by the X chromosome
- they bind and activate “early” promoter (Pe) of Sxl gene in females only
- this promoter generates a functional, properly spliced product
- later, Pm is active
Drosophila sex determination by alternative splicing - what is Pm?
- “maintenance” promoter
- it is active in both females and males
- the pre-mRNA from this promoter requires an Sxl protein for proper splicing
Drosophila sex determination by alternative splicing - why is Sxl only present in females
- males only have one X chromosome so the Pe isn’t that active
- so by the time Pm is active (in both males and females) males don’t have a lot of Sxl to properly splice
Drosophila sex determination by alternative splicing - how does Sxl determine sex if its only functional Sxl in females
- Sxl is involved in splicing
- splices both its transcript and Transformer (Tra)
Drosophila sex determination by alternative splicing - how does Sxl work in females
- pre-mRNA undergoes female-specific splicing which skips exon 3
- allows for the production of a functional Sxl protein
Drosophila sex determination by alternative splicing - how does Sxl work in males
- pre-mRNA undergoes default splicing that includes exon 3
- contains a premature stop codon and leading to no functional Sxl protein
Drosophila sex determination by alternative splicing - Transformer (Tra) Activation
- functions as a splicing activator and is essential to downstream gene regulation
- splicing of Tra gene is regulated by Sxl gene
Drosophila sex determination by alternative splicing - Transformer (Tra) Activation in females
- because Sxl is functional, it removes exon 2 from Tra
- produces a functional Tra protein
Drosophila sex determination by alternative splicing - Transformer (Tra) Activation in males
- without Sxl, exon 2 is retained
- causes a premature stop codon, leading to no functional Tra protein.
Drosophila sex determination by alternative splicing - Double sex (Dsx)
- key effector gene controlling sexual differentiation
- regulates target genes that determine sex-specific traits
Drosophila sex determination by alternative splicing - Double sex (Dsx) in female
- since Tra is functional, it modifies Dsx pre-mRNA, producing DsxF (Female doublesex)
- which directs female development
Drosophila sex determination by alternative splicing - Double sex (Dsx) in male
- without Tra, the default splicing produces DsxM (male doublesex)
- which directs male development.
