Splicing Flashcards
Briefly summarise the mechanism of splicing *
Splicing carried out by spliceosome which recognizes specific nucleotide sequences at the 5’ splice site, the 3’ splice site, and the branch point within the intron.
The 2’-OH group of an adenosine residue at the branch point attacks the phosphate group at the 5’ splice site.
This reaction cleaves the 5’ splice site and forms a lariat structure, with the intron looped back on itself.
The free 3’-OH group at the end of the upstream exon attacks the phosphate group at the 3’ splice site.
This reaction joins the two exons and excises the intron as a lariat structure.
What needs to be ensured when splicing
What are the different components of the spliceosome*
Made up of key snRNAs:
U1 snRNP: Recognizes the 5’ splice site.
U2 snRNP: Binds to the branch point sequence and helps position the branch point adenosine.
U4/U6 snRNP: Forms a complex with U5 and facilitates spliceosome assembly. U6 also participates in catalysis by replacing U1 at the 5’ splice site.
U5 snRNP: Aligns the 5’ and 3’ exons for ligation.
SR proteins: Bind to exonic splicing enhancers (ESEs) and promote exon recognition.
Explain the assembly and action of the spliceosome*
U1 and U2 assemble onto pre-mRNA in a co-transcriptional manner
The U1 and U2 snRNPs interact with each other to form the pre-spliceosome (complex A)
the preassembled tri-snRNP U4–U6•U5 is recruited to form complex B
complex B undergoes a series of rearrangements to form a catalytically active complex B (complex B*)
Complex B* then carries out the first catalytic step of splicing, generating complex C, which contains free exon 1 and the intron–exon 2 lariat intermediate
Complex C undergoes additional rearrangements and then carries out the second catalytic step, resulting in a post-spliceosomal complex that contains the lariat intron and spliced exons
Step 7 – Release of spliced mRNA and lariat (intron)
What is alternative splicing and what is it regulated by *
During alternative splicing, different combinations of exons and/or introns are selected to form mature mRNA. This is regulated by splicing factors (e.g., SR proteins, hnRNPs) and sequence elements like exonic/intronic splicing enhancers (ESE/ISE) or silencers (ESS/ISS).
How can mRNA transcript variation be produced*
Exon skipping: Certain exons are skipped in some mRNA variants.
Mutually exclusive exons: One of two exons is included in the transcript.
Alternative splice sites: Different 5’ or 3’ splice sites are used.
Intron retention: An intron is retained in the transcript instead of being removed.
How can introns act as a method of regulation
What can splicing determine
What are the different types of alternative splicing
How can different 5’ ends be generated by alternative splicing *
Generated through alternative transcriptional start sites that can promote alternative splicing (Different transcription start sites within a gene can lead to the production of transcripts with alternative 5’ ends.)
A gene may contain multiple alternative first exons, each with its own promoter or transcription start site.
These exons can be included in the transcript depending on:
Tissue-specific or developmental-stage-specific expression.
How can different 3’ ends be generated by alternative splicing *
When different poly(A) sites are utilised for transcriptional termination
• Different use in different tissues
• Alternate splicing can occur when different poly(A) sites used (Different polyadenylation signals (AAUAAA) within the same pre-mRNA transcript can be used, leading to the generation of different 3’ ends.)
How can different middle portions be generated by alternative splicing *
Tissue-specific splicing factors acting on the pre-mRNA
certain internal exons are skipped in some transcripts and included in others.
Different tissues express distinct sets of splicing factors that regulate alternative splicing in a tissue-specific manner. These splicing factors interact with cis-acting regulatory elements in the pre-mRNA to control which exons are included or excluded.
What is exon shuffling
How can exon shuffling occur*
Non-homologous recombination involves the formation of DNA double-strand breaks (DSBs) in different exons or gene regions.
These breaks can occur spontaneously or be induced by factors like DNA damage, transposon activity
The broken ends of DNA sequences are then joined, but the joining happens without sequence homology between the recombining fragments.
What are tissue specific splicing factors *
Tissue-specific splicing factors are proteins that regulate alternative splicing in a manner that is specific to particular tissues, organs, or developmental stages. These factors control the inclusion or exclusion of specific exons in the mRNA transcript, thereby generating different protein isoforms tailored to the needs of a particular tissue. By binding to regulatory sequences in the pre-mRNA (such as exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), or intronic splicing enhancers (ISEs)), tissue-specific splicing factors can promote or inhibit splicing events, resulting in different splicing patterns in various tissues.
How does drosophila sex determination work with reference to Sxl *
key player in this process is the Sex-lethal (Sxl) gene
Once Sxl is activated, it regulates the alternative splicing of several downstream genes involved in sex determination, most notably Transformer (tra) and Doublesex (dsx).
Sxl activates the splicing of the tra pre-mRNA to produce a functional TRA protein. This protein is crucial for the female-specific splicing of the doublesex (dsx) gene.
In males, the lack of Sxl leads to non-functional TRA protein, and the doublesex gene is spliced differently, generating male-specific isoforms.
What is the effect of the absence / presence of Tra in males and female drosophila
In females, TRA (from the activation of Sxl and tra) promotes the inclusion of female-specific exons in dsx, leading to the production of the female-specific DSX-F protein.
In males, the absence of TRA leads to the production of DSX-M. The DSX-F and DSX-M proteins then bind to different sets of target genes, driving the development of male and female phenotypes.
What is the role of dsx gene in drosophila*
dsx is a sex-specific transcription factor that mediates the development of male and female phenotypic traits.
The two isoforms of Doublesex (DSX-F and DSX-M) function as transcription factors, binding to specific DNA sequences and regulating the expression of target genes that are responsible for sexual dimorphism.
DSX-F activates genes required for female-specific development and represses male-specific development.
DSX-M activates genes required for male-specific development and represses female-specific gene expression.
First one is male symbo, second is female
What is the role of fru gene in drosophila*
The fruitless (fru) gene in Drosophila is a critical gene involved in sex-specific behavior, particularly in male courtship behavior.
What are SR proteins and their role in regulating splicing *
What is the link between splicing and transcription
What is the intron early theory *
This theory suggests that introns were present early in the evolutionary history of life, even in the earliest forms of life, and that their role evolved over time
introns were a feature of the common ancestor of all life, even before the divergence of prokaryotes (bacteria and archaea) and eukaryotes. According to this theory, early genes in both prokaryotes and eukaryotes contained introns, which were later lost in the lineage leading to prokaryotes.
What is the intron late theory
The Intron Early Theory is contrasted with the Intron Late Theory, which argues that introns were inserted into eukaryotic genes after the divergence of prokaryotes and eukaryotes, possibly through processes like horizontal gene transfer or retroposition. In this view, prokaryotes initially had intron-free genomes, and introns were later added to eukaryotic genes as part of the evolutionary process.
What is LECA*
LECA stands for the Last Eukaryotic Common Ancestor, which refers to the most recent common ancestor of all modern eukaryotes. It is the hypothetical single-celled organism from which all eukaryotic life forms (plants, animals, fungi, protists, and others) evolved.
