RNA quality control in eukaryotes and their implication for health and disease Flashcards
Vorlesung 11
Nenne vier mRNA defekte, welche zum mRNA Abbau führen
vorzeitiges Stopcodon (NMD)
aufgehaltene Translation durch Sekundärstruktur (NGD)
Translation in 3’UTR (REMD)
kein Stopcodon (NSD)
Nenne die Schritte der mRNA Prozessierung
Ausgangspunkt: Primärtranskript
- 5’ Cap via Cap Protein
- 3’ Polyadenylierung
- pre mRNA splicing
- mRNA Splicing
- Export
1 und zwei zwingend notwendig für (u.a. Export-) kompetente mRNA
Weswegen liegen Gene in der Form von Exons, unterbrochen von Introns vor?
a) Larger genetic variability
1. multiple proteins from one gene (via alternative splicing)
2. new genes by exchange of exons
3. post-transcriptional regulation of gene expression
b) Increased stability
I. Short exons will remain more likely intact with regard to recombination events
c) exons often coding for functional domains
Wie viele Exons und Introns enthält pre-mRNA im Schnitt?
7-8 Introns, 8-9 Exons
Nenne drei konservierte Sequenzen in Exons/Introns, welche zum Splicing notwendig sind
- AG
- GU
AG/GU: 5’exon/intron, 3’ intron/exon junction - Branch Point Nukleotid A
Beschreibe die zwei Schritte vom typischen Intron Removal
*Step 1:
an A-residue in the branch point sequence carries out a nucleophilic attack on the 5‘ splice site, creating lariat-exon. The splicing intermediates are: exon 1 and lariat-exon 2.
*Step 2:
exon 1 attacks the 3‘ splice site to generate the splicing products spliced exon and lariat intron.
Was ist alternatives Splicing und wozu ist es gut?
Alternative splicing: is a process by which a single transcript yields different mature mRNAs leading to the production of protein isoforms with diverse or even antagonistic functions
Wie viele mRNAs in Menschen sind, schätzungsweise, alternativ gespiced?
Wie viele davon enthalten vorzeitige Stopcodons (PTC)?
- ca 74%
- Davon ca. 30% (Insgesamt ca.14,7%)
Nenne sechs konservierte RNA Sequenzen, die in die Spliceregulation involviert sind
- branch point (BP, U2 binding site)
- the 5‘ splice site (U1 binding site)
- the 3‘ splice site (U2AF heterodimer binding site)
- splice sites of an alternatively spliced cassette exon (blue box)
- exonic and intronic splicing enhancer (ESE, ISE)
- silencer (ESS, ISS) elements
Welche Auswirkungen haben Punktmutationen in den spliceregulatorischen Sequenzen?
Verantwortlich für 9-10% der genetischen Erkrankungen, die durch Punktmutationen hervorgerufen werden
Nenne die zwei Ebenen des splicing codes
The first layer of the splicing code consists of consensus splice site sequences positioned at exon-intron junctions (AG/GU/Branch Point)
second layer of the splicing code (ESE, ESS, ISE, ISS) directs splicing machinery to the appropriate sites and prevents the usage of cryptic splice sites
Wie viele Mutationen im Menschen betreffen die splice sites (in %)?
ca. 10%
Wie wird das alternative Splicing reguliert?
dynamische Interaktion zwischen hnRNP proteinen ( binden an ESS und ISS) und SR Proteinen (binden an ESE und ISE)
Welche Konsequenz kann die Störung des Antagonismus zwischen SR und hnRNP haben?
weitreichende Folgen. Resultat: genetische Erkrankungen
Wie kann Splicing in Kranklheiten involviert sein?
Splicing can
*be the direct cause of the disease
*modify the severity of the disease
*determine the disease suseptibility
Was ist der Unterschied zwischen Störungen in Cis-agierenden und Trans-agierenden Faktoren?
Cis: Effekt in Cis; nur ein Gen betroffen
Trans: Effekt in trans; potentiell mehrere gene betroffen
Nenne drei Mutationen, die das Splicing beeinflussen, sowie Beispiele für Erkrankungen verursacht durch diese.
Gain-of-splicing-function mutation:
If a splicing element is enhanced or created (creation of cryptic splice site, ESE, ESS, ISE and ISS elements)
Exon 3 skipping in familiar isolated growth hormone deficiency (IGHDII)
Lost-of-splicing-function mutation:
If a splicing element is weakenedor destroyed (e.g. disruption of an ESE or ESS)
* beta-thalassemia: creation of cryptic 3‘ splice site in the first intron
* Spinal muscular atrophy (SMA)
Mutations and alterations of splicing factors:
If a splicing factor for constitutive or alternative splicing is mutated or nongenetically altered (e.g. PRPF3, PRFP8 and PRFP31 in retinitis pigmentosa)
Was ist Spinale Muskelatrophie?
Mutation in SMN (Survival of Motor Neuron)-Gen -> Verlust der Motorneuronen in der Wirbelsäule -> muskeln nonfunktional
Nenne vier Krankheiten, die durch Mutation im Cis-agierenden Splice Code ausgelöst werden
SMA, Becker/Duchenne Muskeldystrophie, frontotemporale Demenz und cystische Fibrose
Was Verursacht SMA?
Gain-of-ESS-function mutation: important in determining the severity of spinal musclar atrophy (SMA)
- SMA is caused by homozygous lost of the survival of motor neurons 1 (SMN1) gene; SMN protein is required for the assembly of the core snRNPs in the cytoplasm
- Severity of SMA is modified by the production of SMN protein encoded by the paralog SMN2
- C-to-U transition inactivates a ESE and at the same time creates a new ESS that is believed to increase exon 7 skipping (!!)
- smn2 gene is nearly identical to smn1 but carries a C-to-T transition in position 6 of exon 7
- only full-length SMN2 protein is active: therefore the greater the skipping of exon 7 the more severe are the sympthoms among SMA patients
Was verursacht Becker Muskeldystrophie (BMD, milde Form von Duchenne)?
*T/A substitution in exon 31 of the dystrophin gene simultaneously creates a premature termination codon (STOP) and an ESS
*leading to enhanced exon 31 skipping
*This mutation causes a mild form of the Duchenne muscular dystrophy disease (called BMD) because the mRNA lacking exon 31 produces a partially functional protein.
welche Mutation verursacht frontotemporale Demenz (FTDP-17) und wieso?
*Mutations within exon 10 of the MAPT gene encoding the Tau protein affect splicing regulatory elements and disrupt the normal 1:1 ratio of mRNAs including or excluding exon 10.
*perturbed balance between Tau proteins containing either four or three microtubule-binding domains (4R-Tau and 3R-Tau), causing the neuropathological disorder FTDP-17.
*One example is the N279K mutation, which enhances an ESE function, promoting exon 10 inclusion and shifting the balance toward increased expression of the 4R-Tau protein variant.
Nenne die Ursachen von cystischer Fibrose
*Cystic fibrosis transmembrane conductance regulator (CFTR) gene exon 9 exhibits slight exon skipping even from the normal allele.
*Polymorphic (UG)m(U)n tracts within the 3’ splice site of the CFTR gene exon 9 influence the extent of exon 9 inclusion and the level of full-length functional protein.
*The severity mutations elsewhere in the CFTR gene is modulated by the level of exon 9 inclusion (mild vs. severe form of CF).
*Individuals with longer, mutated (UG)m(U)n tracts exhibit more exon 9 skipping in part through binding of TDP-43 protein.
Wodurch wird Retinitis Pigmentosa ausgelöst?
Mutation in PRPF31, PRPF8, PRPF3- pre-mRNA- processing factor genen (Notwendig für U4.U5.U6 tri-snRNP assembly des Spliceosomes
Resultat: Aberrantes Splicing von Photorezeptorgenen -> Verlust von Photorezeptoren
