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.
What are the roles of introns during life phases
What is meant by genomic introns*
Introns can play a role in controlling when and how genes are expressed. They contain regulatory elements such as enhancers, silencers, and splicing sites that influence the transcription process. These regulatory elements can be bound by specific proteins or transcription factors, thereby influencing which genes are transcribed, how much mRNA is produced, and when it happens.
What are the different functions of the genomic intron *
Can have a sequence that allows binding of TF (41.2% of TF binding stores found in genomic introns)
Can have alternative transcription initiation sites in introns
Transcription termination sites can be found in them
Can contain protein coding genes
What is meant by transcribed introns*
Transcribed introns refer to intron sequences that are included in the initial primary RNA transcript (pre-mRNA) produced by transcription.
What is a defining characteristic of HES7 gene expression
How might introns impact on oscillating gene networks?*
Introns can have significant impacts on oscillating gene networks—a set of genes that exhibit periodic or rhythmic expression patterns.
What happens when you remove all introns from HES7 gene
Removing some introns can change body pattern
What is meant by spliced introns*
different combinations of exons (the coding regions) are joined together to produce multiple mRNA isoforms from a single gen
What can spliced introns do
Splicing factors and spliceosomal components can interact with transcription elongation factors
U1 on spliceosome can bind with TAT-SF1 on RNA pol 2= promoted elongation and transcription of whole gene
*U2 can bind CPSF on RNApol2 (CPSF binds transcription termination sites) - U1tries to inhibit this
What is an excised intron*
An excised intron is the non-coding region of the gene that is removed from the pre-mRNA during RNA splicing, leaving behind a continuous sequence of exons that will be translated into a protein. The excised intron typically forms a lariat structure and is eventually degraded within the cell.
What are mirtrons and what do they do*
Mirtrons are a class of microRNAs (miRNAs) that are derived from intron sequences of protein-coding genes.
mirtrons regulate gene expression at the post-transcriptional level by binding to complementary sequences in the 3’ untranslated region (UTR) of target mRNAs. This binding can lead to the degradation or inhibition of translation of the target mRNA.
What are snoRNA’s*
SnoRNAs (small nucleolar RNAs) are a class of non-coding RNAs that primarily function in the nucleolus, the sub-nuclear structure responsible for the synthesis and assembly of ribosomes. SnoRNAs are involved in the modification of ribosomal RNA (rRNA) and, in some cases, other small RNAs.
What are EJCs and what do they do*
EJCs (Exon Junction Complexes) are multi-protein complexes that play a critical role in mRNA processing, nuclear export, and post-transcriptional regulation. They are deposited on the mRNA during splicing, right after the removal of introns and the joining of exons.
Roles:
1) Nuclear transport
2) Translation activation
3) mRNA localisation
4) nonsense-mediated decay (NMD)
How are EJCs involved in nuclear transport*
EJCs serve as a mark for properly spliced mRNA, signaling that the mRNA is ready for export from the nucleus to the cytoplasm.
Their presence on the mRNA indicates that the transcript has been processed correctly
EJCs interact with the export receptor complex, such as NXF1 (nuclear export factor 1) and NXT1, which are responsible for facilitating the passage of mRNA through the nuclear pore into the cytoplasm
How are EJCs involved in translation activation*
How are EJCs involved in cytoplasmic localisation*
EJCs interact with proteins that are involved in mRNA localization. These proteins include RNA-binding proteins (RBPs), which direct the mRNA to specific subcellular compartments. The interaction between the EJC and these localization factors ensures that the mRNA is delivered to the correct region of the cell.
How are EJCs involved in nonsense mediated decay*
A surveillance mechanism
Main role is to degrade mRNAs containing a premature stop codon
To prevent dominant-negative/gain of function proteins
What do ribosomes do when no early stop codons are present
Summarise EJCs function of transporting mRNA and the pathways mRNA could go down after
What is meant by overlapping genes
What are the two types of overlapping genes
Same-strand overlapping
Different-strand overlapping
What is meant by same strand overlapping
What is meant by different strand overlapping
What is meant by nested/embedded overlaps
What is meant by partial/terminal overlaps
What are the different ways genes can overlap
What is meant by gene phase
What is meant by in phase overlaps
What is Thermus flavus
What are out of phase overlaps
What are the two products of mouse lnk4a/Arf locus
How can genes partially overlap
What is meant by ribosomal frame shift and what does it depend on
Summarise translation
What are the requirements for mRNA for -1 programmed ribosomal frameshifting to occur
When may slippage occur during the translation elongation cycle
How can -1 programmed ribosomal frameshifting occur
Give an example of +1 programmed ribosomal frameshifting +relationship between ODC and OAZ
Give an example of -1 programmed ribosomal frameshifting
What is cleaved to produce the mature HIV virion
What can stem loops do
Why is overlapping genes beneficial
How can overlapping genes be advantageous in terms of genome size for viruses
How are overlapping genes advantageous for mutations and evolution
What are the differences between pro and eukaryotic genomes
Why else may genes overlap
When can antisense transcription have an impact on gene expression
What is the impact of antisense transcription on transcription initiation
What could happen if you have two overlapping genes that are being transcribed at the same time
What is the impact of antisense transcription on post transcription
How can the sense and antisense pairs act in a self regulatory circuits
What is RNA editing
What are the similarities between RNA editing and splicing
What are the differences between RNA editing and splicing
What are kinetoplast
What kind of structures form in kinetoplasts form
What do maxi circles encode
How is the RNA of cytochrome oxidase II edited
What can the insertion or deletion of uridines do
Where is the information coming from
to perform RNA editing on this scale
with such reproducibility?!
What is the main role of mini circles
Minicircles encoded transcripts “guide” the insertion
and deletions of uridines in maxicircle mRNAs
What are the different parts of gRNA
What is the mechanism of trypanosome RNA editing
What is the editosome
How can alternative RNA editing generate protein diversity*
How can alternative rna editing be used to modify mitochondrial activity
Can produce different proteins
Editing can change the 5’ end to make different proteins
Summarise RNA editing in trypanosomes
What are trypanosomes*
Trypanosomes are a group of parasitic protozoa belonging to the genus Trypanosoma
What is the main type of mammalian mRNA editing
How is apolipoprotein B mRNA editing done
Truncated means something that has been cut short or reduced in length or size.
What does RNA editing require
What are the features of ADARs
ADAR binds editing site
What is the under / over-editing of mRNA associated with
How is AMPS glutamate receptor in mammals edited
How are ADAR1 mutants embryonic lethal
What can ADAR 1 down regulation lead to
What can ADAR 2 down regulation lead to
What are the main roles of RNA editing in mammals
Give examples of Higher levels of RNA editing in the
nervous system?
How can RNA editing drive brain evolution
Summarise regulation of RNA editing in mammals
