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What is the evolutionary origin of minor introns?
Complementarity between snRNAs and the splice sites (and branching point) in the intron suggests that the snRNAs of the two types of spliceosomes co-evolved with their respective intron substrates.
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Example of species that lost minor-class introns:
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What some possible features that might make minor-class introns disadvantageous from the evolutionary standpoint?
- longer recognition motifs at the 5’ splice site and branching point (and, hence, higher susceptibility to mutations)
- slower processing rates
- higher splicing error rates
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What is another name for self-splicing introns?
ribozymes! (catalytic-ally active RNA)
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Group I introns are found in ...
Group I introns are found in mRNA, tRNA, and rRNA of plants, lower eukaryotes, and bacteria
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Group II introns are found in ...
Group II introns are found in mRNA, tRNA, rRNA of plant, fungal and protist chloroplasts and mitochondria and in bacterial mRNA
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Group III introns are found in ...
Group III introns are found in chloroplast mRNA of protists
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What does a Group I intron bind to to initiate splicing
The group I intron sequences bind a free G nucleotide to a specific site to initiate splicing
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What does the Group II intron sequence bind to initiate splicing?
- while the group II intron sequences use a specially reactive A
- nucleotide in the intron sequence itself
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Which group of introns form a lariat that is similar to the method found in spliceosomes?
The group II intron sequences forms a lariat and resembles the pathway catalyzed by the spliceosome
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Support for the hypothesis that the evolutionary origin of Spliceosomal introns come from Group II introns:
- Group II and spliceosomal introns both form a lariat structure
- Mutant Group II introns (that are non-functional because of changes in the secondary structure) still splice in the presence of snRNA
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How can a self-splicing intron act as a genomic parasite?
- Group II intron bound to the intron-encoded protein (IEP) reverse splices into one strand of dsDNA target
- IEP binds to and unwinds the target DNA and the intron self-catalyzes the reverse splicing into one DNA strand (aka retrohoming)
- The second DNA strand is cleaved by the En domain of IEP
- The resulting 3’ OH serves as the primer for reverse transcription of the intron RNA by IEP’s RT domain
- Intron insertion is completed by host-encoded DNA repair processes
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Retrohoming
The major mobility event of group II introns is homing (or retrohoming), in which the intron inserts site-specifically into intronless sites.
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What is "Alternative splicing"?
- a mechanism to generate different RNA products from a single gene
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What are some examples of alternative splicing outcomes?
- Stable versus unstable RNA products
- Secreted versus membrane-bound proteins
- Active versus inactive proteins
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Alternate splicing of mammilian kinase gene: CaMKIIδ
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What are different types of alternate splicing patterns?
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Example of positive regulation of alternate RNA splicing:
- Positive control, in which the splicing machinery is unable to efficiently remove a particular intron sequence without assistance from an activator protein.
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Example of negative regulation of alternate RNA splicing:
- Negative control, in which a repressor protein binds to the primary RNA transcript in tissue 2, thereby preventing the splicing machinery from removing an intron sequence
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What is an Exonic splicing sequence?
- ESE/ESS
- A sequence that can either enhance or silence the inclusion of an exon during RNA processing
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What is an Intronic splicing sequence?
- ISS/ISE
- A sequence that can either enhance or silence the inclusion of an intron during RNA processing
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How is sex determination achieved in Drosophilia?
- sex determination is achieved by a balance of female determinants on the X chromosome and male determinants on the autosomes
- one X=male
- two X= female
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How are males determined in Drosophilia?
If there is only one X chromosome in a diploid cell (1X:2A=0.5), the fly is male (as in a typical XY male)
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What are the sex determining chromosomes in Drosophilia?
flies have either one or two X chromosomes and two sets of autosomes
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How are females determined in Drosophilia?
If there are two X chromosomes in a diploid cell (2X:2A=1), the fly is female (as in a typical XX female)
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define aneuploid
How is sex determined in aneuploid Drosophillia?
- aneuploid=having an abnormal number of chromosomes in a haploid set.
- In aneuploid flies, sex is still determined by the X/A ratios.
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Three genes that are involved in the sex determination pathway of the fruit fly
- SXL = Sex-Lethal (splicing repressor)
- TRA = Transformer (splicing activator)
- DSX = Doublesex (transcription repressor)
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What is the X chr/Autosome ratio in Drosophilia that results in male differentiation pathway development?
0.5
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What is the 'default' sex pathway in Drosophilia?
Male is the “default” pathway in which the Sxl and Tra genes are both transcribed, but the RNAs are spliced constitutively to retain an exon with a premature stop codon
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In Drosophilia, what happens in the absence of a functional Tra product?
In the absence of functional Tra product, Dsx transcript is spliced to produce a protein that turns off the genes that specify female characteristics
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What is the X chr/Autosome ratio in Drosophilia that results in female differentiation pathway development?
1
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Describe how the Sxl gene/protein is used to ensure expression in females:
- In females, Sxl gene transcripts are spliced (and translated) to give rise to functional Sxl protein, an RNA-binding splicing regulator (a repressor!)
- Sxl protein autoregulates its own splicing (to ensure its expression in females)
- Sxl enforces alternative splicing of the Tra transcript (by blocking the 3’ splice site) -> active Tra regulatory protein
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How does the Tra gene/protein act in the Drosophilia sex determination pathway?
- The Tra protein is an activator of splicing: it binds to specific RNA sequences in an exon and activates a normally suboptimal splicing signal
- The Tra protein, along with a constitutively produced protein, Tra-2, acts to produce a female-specific spliced form of the Dsx transcript
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Describe how Sxl enforces alternative splicing of hte Tra transcript
- In males, splicing factor U2AF binds to a polypyrimidine tract in the 3’ end of intron and promotes splicing at the proximal site of the 2nd exon => male Tra mRNA contains an early stop codon
- In females, SXL (a splicing repressor) binds to the polypyrimidine tract, thus blocking access of U2AF to this splice site; U2AF binds and promotes splicing at an alternative distal 3’ splice site => female functional Tra protein
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What is the result of Trans-splicing?
- Results in a production of a chimeric RNA molecule transcribed from:
- two distantly located genomic loci, or
- from the opposite DNA strands (sense and antisense) of the same locus, or
- from the maternal and paternal copies of the same locus
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What is Trans-splicing?
A type of RNA processing in which 5’ and 3’ ends of mRNA come from two different transcripts
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Describe intragenic trans-splicing
- Intragenic trans-splicing can generate mRNAs containing tandem duplications of specific exons (dark blue)
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Describe intergenic trans-splicing
- Intergenic trans-splicing generates chimeric mRNAs (grey and light blue) between pre-mRNAs originating from two different genes
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SMaRT technology
Spliceosome-mediated RNA trans-splicing (SMaRT) technology to reprogram genes for diagnostic and therapeutic purposes
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Some examples of SMaRT applications:
- correction of mutated genes
- suicide gene therapy to express toxic peptides in tumor cells
- expression of therapeutic proteins in animals and humans
- reporter-gene expression
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Mutations in some non-essential components of the major or minor spliceosomes result in tissue-specific defects. Why do you think that is the case?
- If a specific tissue is defect, that tells you that the factor that is
- responsible for that phenotype has activity in that tissue that is not redundant with other similarly functioning or sequence related factors.
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What would you need to do to convert a minor (U12) intron in its native genomic context to a major (U2) intron (or vice versa)? What do you think the outcome of that change be on the phenotype of the organism?
- To convert beteween major and minor introns, you could do point mutations to do the conversion, like Crispr cas9
- Minor-class introns harbor variant but highly conserved 5’-splice sites and branchpoints
- Minor splice sites would need to be converted to major splice sites, e.g., via prime editing
- The rate of splicing will increase, and the tissue specificity of splicing may be compromised, so more mature mRNA may form and in tissues that normally may not have much of that RNA -> overexpression or ectopic expression -> potential phenotypes
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What are some possible benefits of having minor class introns?
- Minor introns (or the minor splicing machinery) make a good potential target for the post-transcriptional regulation of gene expression, because the splicing of minor introns is inefficient (and thus represents a rate-limiting step in the processing of the pre-mRNAs that harbor them)
- In fact, minor-type introns are found in genes that carry out essential functions (e.g., DNA replication and repair, transcription, RNA processing, and translation).
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What are some disadvantages of minor-class introns?
- longer recognition motifs at the 5’ splice site and branching
- point (and, hence, higher susceptibility to mutations)
- slower processing rates
- higher splicing error rates
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Evidence for the evolutionary origin of spliceosomal introns:
- Group II and spliceosomal introns both form a lariat structure
- Mutant Group II introns (that are non-functional because of changes in the secondary structure) still splice in the presence of snRNA
- The reverse is also true: some secondary structure domains of a Group II intron can substitute snRNA of the spliceosome
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Self-splicing introns as genomic parasites: which group uses 'homing endouclease'?
- Group I intron-encoded homing endonuclease makes a double-strand cut in the recipient DNA molecule
- dsDNA break triggers DNA repair
- During break repair by homologous recombination, the parasitic element (the intron) gets copied into the previously “empty” allele
- This “invasion” of uninfected alleles leads to super Mendelian inheritance of the parasitic intron
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Self-splicing introns as genomic parasites: which group uses 'intron-encoded protein'?
- Group II intron bound to the intron-encoded protein (IEP) reverse splices into one strand of dsDNA target
- IEP binds to and unwinds the target DNA and the intron self-catalyzes the reverse splicing into one DNA strand (aka retrohoming)
- The second DNA strand is cleaved by the En domain of IEP
- The resulting 3’ OH serves as the primer for reverse transcription of the intron RNA by IEP’s RT domain
- Intron insertion is completed by host-encoded DNA repair processes
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In drosophilia, what does the Y chromosome do?
the Y chromosome is not involved in determining sex: rather, it contains genes active in forming sperm in adults
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What is the result of different X:A ratios in drosophilia?
3:2
2:2
2:3
1:2
1:3
- X chromosone : Autosome ratios
- 3:2 Metafemale
- 2:2 Female
- 2:3 Intersex
- 1:2 Male
- 1:3 metamale
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The three genes in the sex determination pathway of fruiflies that are subject to alternate splicing:
- SXL (sex-lethal: splicing repressor)
- TRA (transformer: splicing activator)
- DSX (double sex: transcription repressor)
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