Baumeister DNA/RNA Genetics / Epigenetics

1. DNA-Methylierung (adding of CH3 group at the C-5 in Cytosine e.g.)

2. Chromatin Remodelling / modifications / dynamics

3. non-coding RNAs

• DNA methylation close to the promoter represses transcription (blocking of activator or recruiting of repressor protein)

• Gene expression is associated with mit Demethylation

• Maintenance methylase detects methylated cytosins and also methylates the newly added strand at the right position after DNA replication

• CpG island (only protected CG islands by protein binding are not methylated, methylated CGs may eventually mutate to AT

  • CG patterns can be used to predict biological age of an individual

• in early mammalian developement most methylations are lost -> suppression of maintenance methyl transferase activity and demethylating enzymes

Clusters of CG motifs, typically at strongly expressed genes

Many of those clusters are NOT methylated they are used in bioinformatics for promoter identification

highly conserved and protected regions because otherwise cytosin would have been methylated

If an Allele of a gene is methylated it is inactive if the gene from father is methylated the allele of the mother will be the expressed gene in the offspring

Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin.

—> heterocromatin froming proteins cant “pack” the histones behind that blockade the region stays open as euchromatin

Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes

—> only the gene in the “right” direction is transcribed

Insulators can be Methylated, if the insulator region is methylated and the insulator cant bind and another gene can be expressed (Beckwith Wiedemann Region) this is the case in the Wilms Tumor: IGF2 activates, H19 inhibits cell growth a methylated insulator site allows IGF2 to be transcribed because the Insulater CTCF cant bind anymore and the enhancer now acts on the IGF2 gene, if that is the case on the mother and father allele only IGF2 is transcribed and the cell growth is hyperactive —> cancer

Methylation of DNA and Histones can cause the nucleosomes to pack tightly together

Acetylation of Histones result in loose packing —> TF can bind and genes become active

• miRNAs or ncRNA represent small RNA molecules (22 bp), they bind to 3´end of mRNA so that cant be tranlated, these small RNAs play a role in cell differentiation, growth, mobility and apoptosis

• RNA molecules can also modify chromatin structure and silence transcription by recruiting DNA methyltransferases and specific DNA sequences

• RNA molcules act as regulators, genome stabilisators and defenders against foreign genetic elements

Both are eukaryotik transposable elements

Transposons move by means of a DNA intermediate

Retrotransposons move by means of a RNA intermediate

Transposons:

• contain the transposase gene (element for their transposition)

• cut and paste mechanism or copy and paste

• can move exons from one gene to another

• can move crossing over sites and cause unequal recombination in meiosis

• insertion of transposable element in gene or regulatory sequence can block protein production or increase it

  • new splice sites can be created (alternative splicing)

  • new gene groups or genes can be relocated

  • epigenetic regulation, transcription elongation effects, sense and antisense promotor effect, RNA editing

• LINE-1 most frequent transposon in human

• mutations (SNPs) —> in regulatory elements or in protein coding sequence

• duplications (and mutations additionally = divergence —> Globin gene family) (whole genome duplication —> fruit)

• rearrangements (exon shuffeling (+duplication) or transposition of mobile genetic elemetns = transposons and retrotransposons)

• new genetic material (horizontal gene transfer [bacterial sex pilus] or via Viruses —> DNA Viruses Herpes simplex or Epstein-Barr or RNA Viruses Influenza, Polio)

• Cross over in meiosis (unequal cross over caused by transposable elements that provided more sites for cross-over —> one chromosome with deletion other with addition)

• 
  

10. Gene density differs dramatically on various chromosomes

9. Humans have mucher fewer genes than expected

8. Human genes make considerably more proteins (alternative splicing)

7. Human genes are built more complex than that in other organisms

6. more than 200 genes have no relatives in multicellular organisms, BUT: in bacteria!This may indicate horizontal gene transfer

5. Repetitive DNA dates back more than 800 mio years (fossil recordings)

4. Junk DNA has (may have) important functions

3. Mutation rate in males is 2x that of females. Males introduce more mutations in the gene pool, but are also the motor of evolutionary progress.

2. Humans are 99.9% identical. Most of our genetic differences are also representedin distinct ethnic groups (so called „races“). There is NO scientific basis for a categorization of humans into „races“.

1. These data highlight the importance of free, unlimited access to genome sequences

• The bulk of the human genome is made of repetitive sequences and other noncoding DNA

• only 1,5 % are protein coding exons

• Non-coding DNA contains Pseudogenes, repetative DNA (transposable elements and their related sequences)

• only changes to the germ line are passed on to progeny

• SNPs (mutations) are caused by failures in copying and repair mechanism

• Mutations can change gene regulation (not only gene itself)

• DNA dublications (and divergence) give rise to families of related genes (Globin family)

• whole genome duplications

• exon shuffeling

• mobile genetic elements

• horizontal gene transfer (many genes in human are not fund in other animals but in bacteria)

mRNA / rRNA / tRNA / snRNA (splicing) / snoRNA (assist rRNA generartion) / miRNA (silence and degrade mRNA) / siRNA (turn off gene expression) / piRNA (germ line protect)/ lncRNA (scaffold, regulatory)

a. Expression of Tth Cas9 in C. elegans Intestinal Cells:

• Promoter Selection: Choose a tissue-specific promoter that is active in the intestine of C. elegans. The intestine-specific promoter could be derived from a gene known to be expressed exclusively or predominantly in the intestinal cells, such as the promoter of the vha-6 gene.

• Cloning: Clone the Tth Cas9 coding sequence downstream of the selected intestine-specific promoter in a suitable vector for expression in C. elegans. Include transcriptional and translational regulatory elements for efficient expression.

• Transformation: Introduce the plasmid containing the Cas9 expression construct into C. elegans intestinal cells using microinjection or other established methods for genetic transformation.

b. Additional Steps for CRISPR/Cas9 System:

• sgRNA Design: Design a single-guide RNA (sgRNA) specific to the target mRNA sequence of daf-2. The sgRNA guides Cas9 to the target site, allowing the RNA cleavage to occur.

• sgRNA Expression: Express the sgRNA under the control of a suitable promoter. Often, a U6 promoter or other RNA polymerase III promoters are used for sgRNA expression.

• Co-Transformation: Co-transform C. elegans with both the Cas9 expression construct and the sgRNA expression construct.

c. Monitoring Efficacy of Cas9 Function:

• Phenotypic Analysis: Monitor for phenotypic changes associated with the disruption of daf-2 mRNA. In the case of daf-2, this could include observing alterations in the dauer formation or other phenotypes related to the insulin/IGF-1 signaling pathway.

• Molecular Analysis: Perform molecular assays, such as RT-qPCR, to quantify the expression levels of the daf-2 mRNA. A decrease in daf-2 mRNA levels would indicate the efficacy of the Cas9-mediated RNA cleavage.

• GFP Reporter System: Introduce a GFP reporter system downstream of the daf-2 promoter. If the Cas9-mediated cleavage is successful, it should result in the loss or reduction of GFP expression, serving as an indirect indicator of Cas9 activity.

a. Interpretation of the Result: The inconspicuous phenotype in the F1 generation suggests that there is complementation between the daf-2 [Daf-c (dauer constitutive)] and daf-16 [Daf-d (dauer-defective)] mutations. Complementation occurs when mutations in different genes (in this case, daf-2 and daf-16) restore the wild-type phenotype when present together in the same organism. This could imply that the mutations in daf-2 and daf-16 affect different steps or components within the same pathway, or that they are in genes functioning in parallel or converging pathways.

b. Expected Genotypes in the F1 Generation: Assuming complementation, the F1 generation would be heterozygous for both daf-2 and daf-16 mutations. The genotypes of the F1 progeny would be DdFf, where D represents the wild-type allele (in table +), d represents the daf-2 mutation, F represents the wild-type allele of daf-16 (in table +), and f represents the daf-16 mutation.

c) Segregation of Phenotypes in the F2 Generation:

• Wild type animals (DdFf): Approximately 9/16 or 56.25%

• Dauer-constitutive animals (ddFf or Ddff): Approximately 3/16 or 18.75%

• Dauer-defective animals: Approximately 4/16 or 25% !the epistatic double mutant included

d) The daf-2 mutation likely results in a Daf-c phenotype, and the daf-16 mutation results in a Daf-d phenotype. In the double mutant (Daf-c Daf-d), the two mutations might be epistatic to each other, meaning that the presence of one mutation masks the effect of the other. (the two mutated genes are non allelic) —> the dauer defective mutant might dominate?

• RNA secondary/tertiary structure

• formed by base-pairing between signal stranded region of RNA in a hairpin loop with nucleotides elsewhere in RNA chain

• they interact and can intertwine and by that the reading frame can be changed (supression of termination, frameshifting)

• RNA-polymerase II transcribes pre mRNA (on template strand 3´-5´)

• 5´-mRNA end emerges first and is capped by capping proteins that are linked to phosphorylated C-terminal tail of RNA polymerase

• at the end of transcription the 3´end is polyadenylated (3´end processing proteins on tail)

• still in the nucleus the pre-mRNA is spliced (splicing proteins also on the tail) (introns marked by consensus sequences), intron form lariat and the two remaining exon ends are ligated always an free -OH group as transfer element, many RNA-protein complexes (U1-6 snRNPs) are involved, splicosome

• alternative splicing can be tissue specific

• self splicing intron do not need proteins (=ribozymes) e.g. in UPRer the Ire-1 protein splices a pre mRNA to generate a mRNA that translates to TF for stress response genes

• Self‑splicing occurs by a phosphoester transfer mechanism The 3'‑OH of the guanine nucleotide is the nucleophile that attacks and joins to the 5' phosphate of the first nucleotide of the intron

• translation

• silencing or degradation?

• P-body (Cytosolic structure, involved in mRNA degradation)

• stress granule (cytosolic foci, consist of proteins, (m)RNAs (and lipids), stress induced, help to stabilize mRNAs)

• through NPC (nuclear pore complex)

• regulated by nuclear export signal and receptors

• RNA binding proteins

• RNA processing and quality control

• normally only processed mRNA can be exported —> HIV virus DNA codes for mRNA that can be fully spliced and exported and then synthesizes a protein that enables the export of the whole viral mRNAs so the virus can reproduce

• cellular signaling and stress can impact nuclear export

The nucleolus is a membrane-less organelle found within the cell nucleus of eukaryotic cells. It is a prominent structure that plays a crucial role in the synthesis of ribosomal RNA (rRNA) and the assembly of ribosomes, which are cellular structures responsible for protein synthesis.

The nucleolus is not surrounded by a membrane, unlike the nucleus itself. It is formed around specific regions of chromosomes called nucleolar organizer regions, where multiple copies of rRNA genes are located.

—> building of the ribosome subunits from rRNAs

• Antibiotic binding the ribosome subunits: tetracycline, spectinomycin …

• Initiation Factors: The initiation phase is a critical regulatory point in translation. Initiation factors, such as eIF4E, eIF4G, and eIF2, play essential roles in recruiting the ribosome to the mRNA, positioning it correctly, and ensuring the proper start of translation. Various signaling pathways, including the mTOR (=translation active)pathway, can influence the activity of initiation factors.

• mRNA Cap Structure: The 5' cap structure of mRNA is essential for translation initiation. The cap-binding complex, eIF4E (4E binding protein blocks translation in heat shock by dephosphorylation of 4E-BP), recognizes and binds to the cap, facilitating the recruitment of the small ribosomal subunit and the initiation of translation.

• RNA Secondary Structures and UTRs: The untranslated regions (UTRs) of mRNA, particularly the 5' and 3' UTRs, can contain regulatory elements that influence translation. Secondary structures in these regions may impede or facilitate the binding of translation initiation factors, affecting the efficiency of translation.

• RNA-Binding Proteins (RBPs): RBPs interact with mRNA molecules (or other way around) and can either promote or inhibit translation. For example, some RBPs bind to the 3' UTR and regulate translation by influencing mRNA stability, localization, or the accessibility of the mRNA to the translation machinery. —> characterizetion by cross-linking (protein to RNA), digestion, immuno precipitation (collect the proteins = antibody specific), analyse by high throughput sequencing (+ separating by SDS), alternative RNA Affinity Chromatography = RNA immobalized to catch the RBPs or mass spectrometry

• MicroRNAs (miRNAs) and Small Interfering RNAs (siRNAs): These small RNA molecules can post-transcriptionally regulate gene expression by binding to specific mRNA sequences. When miRNAs bind to the 3' UTR of target mRNAs, they often repress translation or promote mRNA degradation.

• Initiation Codon Context: The nucleotide sequence surrounding the initiation codon (AUG) can influence the efficiency of translation initiation. Certain sequence features, such as the Kozak consensus sequence, can enhance or inhibit translation initiation at specific start codons.

• RNA Modifications: Post-transcriptional modifications, such as m6A (N6-methyladenosine) methylation, can affect mRNA stability and translation efficiency. The dynamic regulation of these modifications contributes to the fine-tuning of gene expression.

• Regulation by Cellular Signaling Pathways: Various cellular signaling pathways, including those involving kinases and phosphatases, can impact translation. For example, the mTOR pathway is a central regulator of translation initiation, integrating signals from nutrient availability, energy status, and growth factors. Regulation of eIF2(b) by GDP bound = inactivation and activated by GEF exchanging GDP to GTP

• Stress Response and Integrated Stress Response (ISR): Cellular stress conditions, such as endoplasmic reticulum (ER) stress or amino acid deprivation, can activate the integrated stress response. This response involves the phosphorylation of eIF2α, leading to global translational repression while allowing the preferential translation of stress-response genes.

• Translation repressors (repressor proteins that bind and block)

• RNA thermosensors (RNA structure opens with increased temperature —> listeria virulence genes)

• small molecules that change RNA secondary structure (S-adenosyl methionin [SAM] blocks synthesis of its own production enzymes)

• antisense RNAi that bind the mRNA (opposite strand -base pairing) and blocks translation, experiments in C. elegans showed that insertion of double strand RNA was more effective to produce interference (because of DICER —> dsRNA cutter to make siRNAs and RISC / Argonaut proteinfamily —> using the siRNAs as guides to cleave the target mRNAs

Iron starvation / Excess iron

ferritin and transferrin mRNA have hairpin before and after coding mRNA

cytosolic aconitase is iron response element bindung protein depending on iron conc —> low iron it blocks translation by binding on hairpin before coding region —> no ferritin but transferrin translated

—> high iron conc. —> iron binds aconitase and releases it —> ferritin translated but transferrin has endonucleolytic cleavage domain in hairpin so transferrin mRNA is degraded

antisense RNAi that bind the mRNA (opposite strand -base pairing) and blocks translation, experiments in C. elegans showed that insertion of double strand RNA was more effective to produce interference (because of DICER —> dsRNA cutter to make siRNAs and RISC / Argonaut proteinfamily —> using the siRNAs as guides to cleave the target mRNAs

the dsRNA can be fed to C. elegans via bacteria and the RNA interference will work through the boundaries of cells

small RNA molecules work as guides for the Cas enzymes to detect the specific complementary sequence and cut the DNA there

• the synthetic guide RNA (sgRNA) is needed to use CRISPR / Cas in synthetic biology it needs to be transcribed together with the Cas protein, together they can bind or cleave the right DNA sequence —> fusing regulatory proteins to Cas and binding by sgRNA to promotor regions can also be a useful tool!