L4: Posttranscriptional regulation of eukaryotic gene expression Flashcards
What is posttranscriptional regulation, and how does it differ between prokaryotic and eukaryotic gene expression?
- control of gene expression at RNA level (after transcription, before translation)
- In prokaryotic cells = DNA is transcribed into RNA, which is then translated into protein in the cytoplasm
- Eukaryotic gene expression = DNA transcribed into pre-mRNA, modified & exported from the nucleus to the cytoplasm
( In the cytoplasm, mature mRNA is translated into protein )
What are some of the diverse posttranscriptional mechanisms in eukaryotic gene expression?
Start of transcription
Possible attenuation where RNA transcript aborts
Capping of non-functional mRNA sequences
Slicing and 3’-end cleavage
Possible RNA editing
Nuclear export followed by retention and degradation in the nucleus
Spatial localization in the cytoplasm, leading to translation blockage
Start of translation, also leading to translation blockage
Possible translational recording
Possible RNA stabilization or degradation
Continued protein synthesis
What is spliceosome-dependent pre-mRNA splicing?
- Pre-mRNA contains UTRs, exons, and introns.
- Spliceosomes = complexes that remove introns from pre-mRNA through splicing.
- Splicing produces mature mRNA by joining exons together after removing introns
What are the core splicing signals present in a typical spliceosomal intron, and what are their functions?
The core splicing signals within an intron are:
- Left (5’) splice site
- Branch point sequence: C - T - A/G - A - C - T
- Polypyrimidine tract
- Right (3’) splice site
these core cis-elements & trans-acting factors are essential for proper splicing and intron excision
Describe the assembly and catalysis of spliceosome during splicing
- spliceosome & associated protein factors are crucial for intron excision
- Small nuclear RNAs (snRNAs) interact with protein factors to form small nuclear ribonucleoproteins (snRNPs).
- spliceosome assembly involves several steps:
1. E complex: Formation of a commitment complex with U1, U2AF, BBP, and SR proteins.
2. A complex: Addition of U2 snRNP base-pairing with the branch site.
3. B1 complex: Joining of U4/6 and U5 tri-snRNPs.
4. B2 complex: Release of U1 and U4, formation of the catalytic center with U6, and interaction of U5 with exons.
5. C1 complex: First transesterification step cleaves the 5’ splice site.
6. C2 complex: Second transesterification step cleaves the 3’ splice site and ligates exons
How does splicing specificity differ between yeast and humans?
- most genes in multicellular eukaryotes have multiple long introns, small fraction of yeast genes contain short introns
- core splice site sequences in yeast have higher information content than in humans.
- Yeast core elements sufficient to recognize authentic introns, in humans additional mechanisms needed due to lower info content
- this offers rich splicing regulation possibilities in higher eukaryotes
What are the categories of RNA-binding proteins (RBPs) involved in splicing regulation, and how do they function?
- SR PROTEINS (Include SRSF1, SRSF2, SRSF3, SRSF11) contain RNA recognition domains (RRMs) & RS domains; interact with exonic splicing enhancers (ESEs)
- hnRNP PROTEINS: (Include hnRNP I, hnRNP AB, hnRNP A, hnRNP H) contain RRMs & related domains; bind introns and exons; interact with splicing silencers
- Tissue-specific AS REGULATORS: Use RRMs, KH domains, Zn-finger domains; bind tissue-specific splicing enhancers/silencers.
Splicing enhancers and silencers interact with RBPs, allowing core machinery to recognize exons and providing regulatory possibilities
What are the mechanisms of activation and repression of splicing by RNA-binding proteins?
- Activation =
positioning of RNA-binding proteins downstream of the exon can activate splicing, while positioning upstream can repress splicing. - Repression =
RNA-binding proteins can compete with core splicing factors for binding intronic splicing silencers (ISS), bind multiple intronic sites - forming repressive complexes, inhibit intron definition by blocking interactions, or inhibit exon definition by blocking interactions across exons.
Activation & repression effects depend on protein recruitment position with respect to regulated exons
What is the difference between constitutive and alternative splicing?
- Constitutive splicing: All exons joined uniformly in mature RNA, regardless of cell type/conditions.
- Alternative splicing: Exon configuration varies based on circumstances
Alternative splicing is widespread in higher eukaryotes, allowing multiple mature RNA products from the same gene.
What are the biological functions of alternative splicing?
- Increased protein diversity: allows single gene to produce multiple protein isoforms with different functions
- Regulation of gene expression: By including/excluding specific exons, alternative splicing can modulate the levels of certain protein isoforms, influencing gene expression regulation
How does sex determination in Drosophila involve alternative splicing of the Sex-lethal gene?
- Female Drosophila: early promoter (E1) is active in XX individuals - this promoter silences exon2 and exon3 of the Sxl gene. mRNA produced includes exons 4 to 8, generating the Sxl protein
- Male Drosophila: early promoter is silent in XY individuals. As a result, exon2 and exon3 are included in the mature RNA, leading to absence of Sxl mRNA and protein
How does sex determination in Drosophila involve late production of the Sex-lethal (Sxl) protein through splicing-controlled positive feedback?
- Sxl pre-mRNA includes exons 2 to 8, with exons 4 to 8 encoding the Sxl protein.
- early promoter (E1) is silenced in late development
- an Sxl mRNA-binding protein binds intronic splicing silencers upstream & downstream of exon 3 (on the Sxl gene). This protein has two RRM domains (and similar in sequence to tissue-specific AS regulators)
- Developmentally late versions of Sxl mRNA are produced from the later promoter (L1), skipping exon 3 and including exons 4 to 8.
- leads to the production of the Sxl protein
How does sex determination in Drosophila involve a splicing switch regulating the production of the Transformer (Tra) protein?
- Tra pre-mRNA includes exons 1 to 4. Exons 2 and 3 are part of the same exon unit and can be either short or long.
- An Sxl mRNA-binding protein binds an intronic splicing enhancer upstream of exon 2 (on the Tra gene) in females.
- Developmentally late versions of Tra mRNA are produced, including only exons 1, 3, and 4, skipping exon 2.
- leads to the production of the Tra protein as exon 1, 3, and 4 produce a complete open reading frame, promoting female-specific development.
How does sex determination in Drosophila involve distinct splice forms of the Doublesex (Dsx) gene?
- Dsx pre-mRNA includes exons 1 to 6, with exon 4 encoding Dsx protein.
- exonic splicing enhancer located in exon 4 (on the Dsx gene) bound by Tra2 and Tra mRNA-binding proteins in females.
- Developmentally late versions of Dsx mRNA produced, including only exons 1, 2, 3, and 4, skipping exons 5 and 6.
- leads to the production of the female version of the Dsx transcription factor (Dsx F), which promotes female-specific developmental genes
Summarize the sex determination mechanisms in Drosophila for females
Female Drosophila (XX):
Early transcription of the Sxl gene.
Sxl continues to be produced later in development.
Production of functional Tra protein.
Production of the female version of the Dsx transcription factor (Dsx F).
Female development
