Gedächinisprotokoll WS22-23

a) mutations in splice sites can cause exon skipping, intron retention, or the creation of cryptic splice sites

1. mutation at the 2cd consensus sequence of an intron will result in splicing out the next exon because the intron will reach until the next fitting consesus sequence —> removal of exons = exon skipping [alternative splicing])

2. mutations at 1st consesus sequence (or both sequences of an intron) will skip the introns removal and leave it in the processed mRNA —> Introns maintained (intron retention) may result in completely different proteins (missense) or in a premature stop codon (nonsense) or in a frameshift

3. mutation that creates a new splice site (cryptic splice sites) in the exon region —> destroys the exon coding sequence but also in intron region —> inclusion of extra sequences from the adjacent regions

b) = group II intron splicing, the introns have ribozyme activity

Three Stages of Splicing:

• Step 1 (Exon Ligation): The group II intron catalyzes a series of transesterification reactions within its own structure. The 2' hydroxyl group of an adenosine within the intron attacks the phosphodiester bond at the 5' end of the intron, resulting in the ligation of the two exonic sequences.

• Step 2 (Intron Circularization): The 3' hydroxyl group of the 5' exon attacks the phosphodiester bond at the 3' end of the intron, circularizing the intron and releasing the ligated exons.

• Step 3 (Exon Cleavage): The circularized intron is then cleaved, releasing the intron and regenerating the active site for subsequent rounds of splicing.

Another event where splicing without a splicosome is occuring is in the unfolded protein stress response from the ER, where the IRE1 protein catalyzes the unconventional splicing of a specific mRNA called XBP1 (X-box binding protein 1). This splicing event involves the removal of a 26-nucleotide intron from XBP1 mRNA which can then be translated to a transcription factor activating UPRer genes.

Additional information + complex phraseing:

1. Splice Site Mutation Resulting in Exon Skipping:

   • Mutation Type: Point mutation or small insertion/deletion affecting the 5' or 3' splice site.

   • Outcome: The mutation may weaken or abolish the recognition of the splice site by the spliceosome, causing the skipping of an exon during RNA splicing. This leads to an altered mRNA sequence, and the translated protein may lack specific functional domains or have an abnormal structure. This can result in a nonfunctional or partially functional protein, impacting cellular processes.

2. Splice Site Mutation Resulting in Intron Retention:

   • Mutation Type: Mutation affecting the branch point sequence or the polypyrimidine tract within the intron.

   • Outcome: The mutation may disrupt the normal splicing process, leading to the retention of an intron within the mature mRNA. This results in an mRNA with extra, non-coding sequences. When translated, this aberrant mRNA can produce a protein with additional amino acids, often leading to a nonfunctional or structurally altered protein. Intron retention can also trigger nonsense-mediated decay, reducing the overall expression of the mutated gene.

3. Creation of Cryptic Splice Sites by Mutation:

   • Mutation Type: Creation of new splice sites within exonic or intronic regions.

   • Outcome: Mutations can generate novel splice sites, either within exons or introns, which can be recognized by the spliceosome during splicing. This can lead to the inclusion of extra sequences from the adjacent regions, resulting in an altered mRNA and a protein with abnormal structure or function. Cryptic splice site mutations can also introduce premature stop codons, leading to the production of truncated and often nonfunctional proteins. The abnormal protein may interfere with normal cellular processes or be subject to degradation.

a)

1. DNA Methylation: the addition of a methyl group to the cytosine nucleotide in a DNA sequence, forming 5-methylcytosine. DNA methylation is often associated with gene repression. Methylated DNA regions can recruit proteins that inhibit gene transcription, effectively silencing the associated genes

   Histone Modification: Although histones are proteins, modifications to these proteins play a crucial role in controlling DNA accessibility and gene expression. DNA wraps around histone proteins to form nucleosomes. Post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination of histones can influence chromatin structure, making it either more open (euchromatin) or more condensed (heterochromatin)

2. mRNA N6-Methyladenosine (m6A) Modification —> addition of a methyl group to the adenosine nucleotide at the N6 position can alter mRNA metabolism, including stability, splicing, and translation efficiency

   methylation degree of the 5´Cap structure

   another possible mechanism could be small interferring RNAs that can also be passed on to next generations, those molecules could then inhibit / silence or alter mRNA structures

3. post-translational modifications on proteins like Methylation, Acetylation, Ubiquitination [Lysin residues] and Glycosylation are considered epigenetic because they involve heritable changes in gene expression or cellular function that are not encoded in the DNA sequence

b)

1. NF-kappaB pathway —> in inactive state bound by inhibitory protein (kappaBI) by various signaling pathways the IkappaB Kinase is activated and Ubiquitinates the inhibitory protein causing its degradation (proteasomal) —> the NF transcription factor is now free to translocate into the nucleus (activating genes for immune responses, inflammation, and cell survival)

2. STAT pathway —> receptors bind ligands and activate receptor-associated Janus Kinases (JAKs), leading to their autophosphorylation, they then recruit the STAT proteins which are phosphorylated and thereby dimerize, in the dimerized form they can translocate into the nucleus where they act as TF for genes for cell growth, differentiation, and immune responses

other ideas for pathways:

• IRE1 (splicing pre mRNA which then produces then TF and translocates into nucleus)

• ATFS1 (accumulates when TOM complex for Mito entry is blocked because of stress response and only then enters nucleus vie its NLS)

• Accumulation of factors appears also in HiF (hypoxia) or ß-catenin

• BMP pathway where R-Smads are phosphorylated, form Co-Smads with other Smad proteins and are thereby released from the receptor complex and can enter the nucleus

a)

the lung precursers: TFs in foregut specification (NKX2.1 and SOX2), WNT26

Nkx2.1 induces seperation from the gut tube -> lung precursor formation

Nkx2.1 accumulates ventrally by upregulation from BMP, FGF and Wnt

Wnt signalling is upregulated by RA (retinoic acid)

Sox2 inhibited by BMP -> accumulates dorsally

Noggin at dorsal side inhibits BMP -> BMP only ventrally

the lung buds emerge from the ventral surface of the foregut endoderm

FGF for bud formation and lung branching, the cardigenic mesoderm adjacent to the lung assists in lung formation with FGF signaling and cell cell interaction

b)

the splanchnic mesoderm surround the emerging lung buds (visceral mesoderm)

—> it forms the airway smooth mucle cells and connective tissue

—> it also plays a major role in the branching mechnaism by FGF signaling at the branching ends and it further assists lung formation through structural support and vesicularization (blood vessels)

c)

repetitive process with self-limiting feedback loops

• Pathways involved: FGF pathway, Wnt pathway, Shh Pathway, BMP pathway

• Fgf10 from mesoderm (Receptor: Fgfr2) at tips of branches induces expression of BMP4 & Shh, Fgf10 is downregulated by Bmp4 & Shh -> limits outgrowth of individual buds, important for the development of adjacent side branches

• Bicoid gradient – source and sink gradient (diffusion) e.g. anterior posterior dev.

• BMP gradient – time resolved inhibitor e.g. dorsal ventral dpp sog

• Dorsal gradient – nuclear translocation signal via active transport e.g. dorsal ventral dev.

A-P in drosophila by Bicoid (anterior) and oscar/nanos (posterior) the mRNAs are transported along cytoskeleton to each pole and then translated to the protein that form the source and diffuses through the embryo (source) it spreads as fas as it reaches from both sources (sink) and form a gradient

D-V pattering in Drosophila by a time resolved inhibitor gradient with BMP, more specific it is the dpp molecule that is a BMP ligand and spreads along the outer cell layers along the dorsal ventral axis, the gradient is formed by a counter gradient coming from ventral by the inhibitor sog, where much sog is dpp cant interact with its rezeptor and is pushed more and more dorsally, in the end on the dorsal side the amnioserosa is located where the most dpp was bound to its receptor, parts of the neural ectoderm come next and mesodermal parts where the reverse gradient from sog overlaped most

a) cell migration, cytotoxic T-cell, cell division, epithelial cell / epithilia (barrier function, stability, direction), organs / developement of organism and prevention of cisease (cancer)

as text:

important for cell migration (where to move), without polarity of the cytoskeletal filaments no directional transport, important for a cytotoxic T-Cell to be able to detect, souround and digest a target, very important for polarized cell division (e.g. stem cells dividing asymmetrically so one stays stem cell and other differentiate) and epithelial cells need polarity for stability, anchoring and directionality to form a barries, these characteristic are also the important one for epithelia as a tissue without polarity our skin would just rip apart because no mechanical stability would be given and the barrier function would be lost if the outer cells would just be turned upside down for the entire organism every cell and tissue is important to do its job for survival if they lose polarity it means disease (e.g. cancer) for the organism, besides that in the developement of the organism many signaling events and organ developements depend very much on polarity

b)

epithelial cell / epithilia (barrier function, mechanical stability, direction/transport)

c)

Polarity proteins provide a molecular machinery that can establish and maintain polar identity of cellular domains

Apical: Crumbs / aPKC / Par-6 / Par-3

Basel: Scribble / Discs-large (Dlg) / Lethal giant larvae (Lgl)

—> regulatory network of positive and negative feedback amplifies and maintains the polarity cues the cytoskeleton is very much in control of polarity

a) No, a prokaryotic promotor is lacking eukaryotic promotor characteristic e.g. the TATA box and other regulatory domains such as enhancers and silencers, instead is has the Pribnow box which binds the sigma factor in procaryotes, that is such a difference that a binding of the eukaryotic transcription complex would not be possible

b) The basel promotor is a small part of the promotor region in eukaryotes right before the coding sequence starts it contains the TATA box if it is translocated upstream it would defenitly alter the very defined promotor region and could destroy transcription complex binding sites, besides it could shift regions that interact with the transcription machinery far more downstream even if 20 basepairs are not much we would need to experimentally proof that this could not effect the whole complex after all. The only reason why it might be possible is that 20 bp upstream from my knowlege there is not (yet) a detected sequence that is drastically to lose but still the shift of 20 bp would cause problems

a)

Ras —> oncogene

EGF-R —> oncogene

Rb —> Tumor-suppressor

BCR-Abl —> oncogene

Brca1 —> Tumor-suppressor

p53 —> Tumor-suppressor

b)

alterations (mutations, transposons, duplications etc.) in the coding sequence

splicing errors

errors in DNA repair

errors in chromosome recombination

virus infections (oncoviruses)

a)

self renewal

proliferation potential

ability to differentiate (before it should be unspecialized)

multipotancy (very rarely pluripotancy but in early developement often) meaning the property to be able to produce daugther cells that can then differentiate into various cell types dependent on whats needed (signal dependent)

protected from environmental damage and mutation provocing substances or radiation …

a)

1. library preparation (fragmentation of the DNA squence that you want to sequence + linking adapters to the DNA ends)

2. cluster generation (give the DNA on the flow plate it will bind via the adapter sequence to the flow plate where the immobilized counter parts are (hybridisation)

3. washing steps and PCR steps for amplification of the DNA molecules

4. Bridge building and again PCR and washing and everything for both direction of the DNA strand

5. sequencing (paralell fluorecent nucleotides bind and the light emission is detected —> billions of signals -> DATA)

6. Data Analysis (statistical libary of all data + comparing with databases etc.)

b) affected individuals are homozygous or compound heterozygous (e.g. Cystic fibrosis (CF) (= Muskoviszidose)

1. inherited by parents who both needed to be carriers of the disease causing allel (the patient had bad luck if both parents where just carriers and not homozygous)

2. he carried just one disease causing allel and by mutation or chromosome silencing or epigenetic mechanisms on the other chomosome that was enough to cause the disease (=compound heterozygous) or mutations during his lifetime on both allels caused the disease (very very rare i guess)

a)

to be localized in each one of the 3 the protein needs an organelle specific localization signal, it also requires transport machinery and trezeptors on the organelle that detact the sequence and last but not least it need translocator / channels that enable transportation of the protein through the membrane often by energy (ATP/GTP) consumption

Differences:

• Aminoacid pattern of the signal sequence (MTS, NLS = basic AA rich and ERSignalSequence = often hydrophopic)

• proteins for signal detection and transmembrane transport in the 3 organelles (ER signal detecting particle and tranlocator) (Mitos signal rezeptor linkted to TOM complex transport outer membrane and TIM through inner membrane) (nucleus has pores where GTP dependent transporters like Ran activly transport the proteins through the nucleus pore complex)

• protein conformation and in case of ER it could be co-translational translocation

b)

1. immobilze the MLS rezeptor protein on a surface and add the AA sequence if it binds to the receptor it will not be in the flow through because it interacts with the rezeptor, then design a washing step where onyl rezeptor bound molecules are washed out and if your AA sequence is in there you know it is likely to be a MLS

1.1. compare to databases! -> many MLS have a distinct patterning and by bioinformatic studies you can already get an idea if it is likly to be an MLS or not

1. use a reporter like GFP and bind it to the sequence you can then actually see (microscope) where the sequence is transported if it always accumulates at the mito membrane it is very likely to be a MLS