how are transcriptome analyses powerful?
- you can look at the genes and the activation of transcription in different samples and compare them between normal and disease states (aka. how it responds to extracellular stimuli)
- help drug creation
- ex. leukemia differentiation
lecture 2 review- use DNA microarray to analyze the transcriptome
how do transcriptome analyses provide a signature of cell state
explain the experiment for AML (acute myelogenous leukemia) disease and how they used the DNA microarray
- conduct the DNA microarray from someone with primary acute myelogenous leukemia (AML) cells
- do the DNA microarray for differentiated neutrophils (normal cells)
- DNA microarray for model cell line of AML (cells that can grow in the lab that looks like the diseased cells)
- Then, DNA microarray for different drugs:
–> you can use this to see if cancer cells can be changed to look like normal cells with certain drugs
–> helps with drug creation
did the AML disease experiment work to generate working drugs
- what is IC50 – connect to it
IC50: Concentration of
compound required to
inhibit cell proliferation
by 50%
- low IC50 means it takes little of a chemical to create the desired effect
- the IC50 was low for many of the drugs they tested !
How does RNA processing differ in prokaryotes and eukaryotes
- in prokaryotes everything happens in the cytoplasm so you can start translation before transcription ends
- in eukaryotes cannot couple the process. transcription in the nucleus, and translation in cytoplasm
- in eukaryotes there are introns and exons which makes the pre mRNA via transcription
- then RNA processing occurs (covalent modifications and removal of intron sequences):
– 5’ capping
– RNA splicing
– 3’ polyadenylation
A Review of RNA Processing: RNA Capping
- what is it
- what complex is needed for the RNA cap to bind
- what are the three functions of RNA capping
- RNA Capping: Addition of a modified guanine nucleotide to the 5’end of pre-mRNA (3 enzymes involved)
- Cap bound by cap-binding complex (CBC)
Functions:
1. Helps in RNA processing and export from the nucleus for translation
2. Important role in translation of mRNAs in the cytosol
3. Protects mRNA from degradation
A Review of RNA Processing: RNA splicing
- what are exons and introns and what sequences do each include
- what are transcribed into RNA
- what is RNA splicing
- what are exons bonded together with
- what are we left with after all this RNA processing
- Recall that eukaryotic genes are made up of coding sequences called exons and
non-coding sequences called introns
(note: exons also include rRNA and tRNA) - Both introns and exons are transcribed into RNA, but the introns are removed by a process called RNA splicing
- exons remain and are stitched together with covalent and phosphodiester bonds.
- afterwards we are left with a 5’ cap in the beginning, a poly-A-tail at the end, noncoding sequences right after (5’UTR) and before (3’UTR), and a coding sequence in the middle that starts with the AUG codon. This results in the protein.
A Review of RNA Processing: RNA Splicing
what is alternative splicing?
- Alternative splicing: Different cells can splice an RNA transcript differently by selectively including or excluding specific exons to make different proteins from the same gene
- ~75% of human genes produce multiple proteins increases coding potential of genomes
A Review of RNA Processing: RNA Splicing
- what is the spliceosome
- what are exon junction complexes (EJCs)
- what does EJCs look like in RNA that is normally sliced vs abnormally sliced?
- Spliceosome: RNA splicing is carried out by an enzyme complex made up of RNA and proteins
- EJCs: Sites of proper splicing are bound by exon junction complexes (EJCs)
➢ Serve as a marker for properly spliced RNA (they are not holding the exons together - those are already covalently bound – the EJCs are just a marker) - abnormal splicing is when an intron may not be spliced out by mistake via the spliceosome. This will not receive an EJC.
how is alternative splicing regulated via negative and positive control (2 for each)
- negative control
- splicing and no regulation
–> spliceosome recognizes the intron and splices it out
- no splicing and regulation
–> repressor binds to intron region
–> repressor protein stops spliceosome from binding and splicing
- positive control
- no splicing and no regulation
–> spliceosome does not recognize the intron thus no splicing (error)
- splicing and regulation
–> activator protein binds to activator region near the intron region and forces the spliceosome to bind and thus splices.
Drosophila Sex Determination via regulation of alternative splicing:
- how do you determine the sex of a fly? how is this different to humans?
- what are the ratios that determine male or female fly
- in humans sex is determined via the sex chromosomes (#23) : XX is female and XY is male
- in flies, we count the number of X chromosomes (IGNORE Y) and divide it by the number of autosomal sets the fly has to find the ratio (humans usually have 22 autosomal sets i think)
Sex determination ratios:
ratio of X chromosomes (X): autosomal sets (A)
X:A = 0.5 Male (Default)
X:A = 1.0 Female
examples for understanding:
if the fly is X:X and has 2 autosomal sets
–> female 1:1 ratio
(2 X chromosomes / 2 autosomal sets = 1 thus female)
if fly XY with 1 autosomal set
–> female 1:1 ratio
(1 X chromosome (ignore Y) / 1 autosomal sets = 1 thus female)
if XX with 4 autosomal sets
–> 0.5 so male
(2 X chromosomes / 4 autosomal sets = 0.5 thus male)
what are the three genes involved in regulating alternative splicing in drosophila sex determination?
what do these genes do?
Three genes involved:
* Sex-lethal: splicing repressor
* Transformer: splicing activator
* Doublesex: Regulates sex gene expression
All 3 genes contain regulated splice sites
How are the three genes involved in alternative splicing for male drosophila sex determination
Sex lethal gene:
- introns are spliced out via spliceosome
- a nonfunctional protein is produced
- No regulated splicing – no activators or repressors are regulating gene
Transformer gene:
- introns are spliced out via spliceosome
- a nonfunctional protein is produced
- No regulated splicing – no activators or repressors are regulating gene
Doublesex gene:
- introns and a bit of the light blue exon region of the gene that varies between males and females are spliced out via spliceosome (alternative splicing - not only introns are spliced out but some of the exons too)
- produces a functional protein which represses female genes and thus codes for a male
- No regulated splicing – no activators or repressors are regulating gene
How are the three genes involved in alternative splicing for male drosophila sex determination
Sex lethal gene:
- a transient event takes place and produces a special sex lethal splicing repressor
- the repressor binds to the sex lethal gene and blocks the spliceosome from making multiple small cuts like in the male gene. Instead the repressor blocks the space between two introns so the spliceosome cuts a 1 huge section encompassing both the introns and the middle part with the exon.
- this produces a functional sex lethal protein and generates a positive feedback loop. The sex lethal protein produced acts again on the gene as a repressor to produce more of the sex lethal protein.
- regulated splicing as a repressor is used
Transformer gene:
- the sex lethal protein produced via the positive feedback loop acts as a repressor on the transformer gene. forces the spliceosome to cut a bigger section around one of the introns. the other intron is further away and thus is spliced like normal.
- this produces a functional Tra protein
- regulated splicing as a repressor is used
Doublesex gene:
- Functional Tra protein works with a side protein (Tra2) to activate the splicing of the intron properly
- produces a functional gene which represses male gene thus codes for female development
- regulated splicing as an activator is used
A Review of RNA Processing: 3’ Polyadenylation
what are the two proteins
- termination in eukaryotes is much more complex than prokaryotes
- Signals encoded in genome
- RNA polymerase transfers
protein complexes to RNA
two main proteins
- CstF (cleavage stimulating
factor)
- CPSF (cleavage and
polyadenylation specificity factor)
how is the poly A tail created
- RNA polymerase II transcribes DNA, reaching a cleavage/polyadenylation signal sequence (AAUAAA).
- The AAUAAA sequence is recognized by CPSF (Cleavage and Polyadenylation Specificity Factor) and CstF (Cleavage Stimulation Factor).
- These complexes bind to the phosphorylated C-terminal domain of RNA polymerase II.
- CPSF and CstF recruit other factors to cleave the RNA at a specific site downstream of the AAUAAA signal.
- Poly(A) polymerase adds approximately 200-300 adenine nucleotides to create a poly(A) tail at the new 3’ end of the mRNA.
- This addition occurs de novo without a template; there are no complementary Ts in this region of DNA.
- The poly(A) tail is bound by poly(A)-binding proteins that protect it from degradation by exonucleases.
- The presence of this tail facilitates nuclear export, translation initiation in the cytoplasm, and overall stability of the mRNA molecule.
