RNA quality control in eukaryotes and their implication for health and disease

Question 1

Nenne vier mRNA defekte, welce zum mRNA Abbau führen

Answer 1

vorzeitiges Stopcodon (NMD)
aufgehaltene Translation durch Sekundärstruktur (NGD)
Translation in 3’UTR (REMD
kein Stopcodon (NSD)

Question 2

Nenne die Schritte der mRNA Prozessierung

Answer 2

Ausgangspunkt: Primärtranskript

1. 5’ Cap via Cap Protein
2. 3’ Polyadenylierung
3. pre mRNA splicing
4. mRNA Splicing
5. Export

1 und zwei zwingend notwendig für (u.a. Export-) kompetente mRNA

Question 3

Weswegen liegen Gene in der Form von Exons, unterbrochen von Introns vor?

Answer 3

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

Question 4

Wie viele Exons und Introns enthält pre-mRNA im Schnitt?

Answer 4

7-8 Introns, 8-9 Exons

Question 5

Nenne drei konservierte Sequenzen in Exons/Introns, welche zum Splicing notwendig sind

Answer 5

1. AG
2. GU
   AG/GU: 5’exon/intron, 3’ intron/exon junction
3. Branch Point Nukleotid A

Question 6

Beschreibe die zwei Schritte vom typischen Intron Removal

Answer 6

*Step 1:
an A-residue in the branch point sequence carries out a nucleophilic
attack on the 5‘ splice site, creating lariat-exon 2. 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.

Question 7

Was ist alternatives Splicing und wozu ist es gut?

Answer 7

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

Question 8

Wie viele mRNAs in Menschen sind, schätzungsweise, alternativ gespiced?

Wie viele davon enthalten vorzeitige Stopcodons (PTC)?

Answer 8

1. ca 74%
2. Davon ca. 30% (Insgesamt ca.14,7%)

Question 9

Nenne sechs konservierte RNA Sequenzen, die in die Spliceregulation involviert sind

Answer 9

• 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

Question 10

Welche Auswirkungen haben Punktmutationen in den spliceregulatorischen Sequenzen?

Answer 10

Verantwortlich für 9-10% der genetischen Erkrankungen, die durch Punktmutationen hervorgerufen werden

Question 11

Nenne die zwei Ebenen des splicing codes

Answer 11

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

Question 12

Wie viele Mutationen im Menschen betreffen die splice sites (in %)?

Answer 12

ca. 10%

Question 13

Wie wird das alternative Splicing reguliert?

Answer 13

dynamische Interaktion zwischen hnRNP proteinen ( binden an ESS und ISS) und SR Proteinen (binden an ESE und ISE)

Question 14

Welche Konsequenz kann die Störung des Antagonismus zwischen SR und hnRNP haben?

Answer 14

weitreichende Folgen. Resultat: genetische Erkrankungen

Question 15

Wie kann Splicing in Kranklheiten involviert sein?

Answer 15

Splicing can
*be the direct cause of the disease
*modify the severity of the disease
*determine the disease suseptibility

Question 16

Was ist der Unterschied zwischen Störungen in Cis-agierenden und Trans-agierenden Faktoren?

Answer 16

Cis: Effekt in Cis; nur ein Gen betroffen

Trans: Effekt in trans; potentiell mehrere gene betroffen

Question 17

Nenne drei Mutationen, die das Splicing beeinflussen, sowie Beispiele für Erkrankungen verursacht durch diese.

Answer 17

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)

Question 18

Was ist Spinale Muskelatrophie?

Answer 18

Mutation in SMN (Survival of Motor Neuron)-Gen -> Verlust der Motorneuronen in der Wirbelsäule -> muskeln nonfunktional

Question 19

Nenne vier Krankheiten, die durch Mutation im Cis-agierenden Splice Code ausgelöst werden

Answer 19

SMA, Becker/Duchenne Muskeldystrophie, frontotemporale Demenz und cystische Fibrose

Question 20

Was Verursacht SMA?

Answer 20

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

Question 21

Was verursacht Becker Muskeldystrophie (BMD, milde Form von Duchenne)?

Answer 21

*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.

Question 22

welche Mutation verursacht frontotemporale Demenz (FTDP-17) und wieso?

Answer 22

*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.

Question 23

Nenne die Ursachen von cystischer Fibrose

Answer 23

*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.

Question 24

Wodurch wird Retinitis Pigmentosa ausgelöst?

Answer 24

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

Question 25

Nenne vier für das 3’-Processing kritische Motive der mRNA

Answer 25

• USE= upsteam sequence element
• AAUAAA= poly(A) signal
• CA= Cleavage site
• DSE= downstream sequence element

Question 26

Nenne die Schritte des 3’-Processings sowie die involvierten Proteine

Answer 26

1. Assembly of multiprotein complexes at specific RNA recognition motifs

Protein complexes involved:
* CPSF= cleavage/ polyadenylation specificity factor
* CstF= cleavage stimulating factor
* CF I= Cleavage factor I
* CF II= Cleavage factor II
* PAP= poly(A) polymerase

2. Cleavage at the cleavage site by CPSF 73
3. • Formation of a poly(A) tail by PAP (addition of ~250 A residues) that is bound by PABP

Question 27

Gibt es RNA, die nicht polyadenyliert wird?

Answer 27

Ja. replikationsabhängige Histon mRNA.

Question 28

Nenne vier medizinisch relevante Fehler im 3’-end-processing, sowie Beispiele für Erkrankungen oder Schäden.

Answer 28

• lost-of function mutation (AAUAAA):
  Alpha-thalassemias (alpha-globin gene)
  Beta-thalassemias (beta-globin gene)
  IPEX syndrome (Fox3p gene)
  Fabry disease (alpha-GalA gene, AAUAAA signal within ORF, no 3’UTR)
• Gain-of function mutation (CA):
  Thrombophilia, prothrombin
  Thrombophilia, fibrinogen
• USE: important for 3’ processing :
  LaminB
  Complement C2
  Cyclooxygenase-2 (Cox-2)
  Collagen
• Alternative poly(A) signal recognition:
  IgM heavy-chain pre-mRNA
  during B-cell differentaition

Question 29

Nenne zwei medizinisch relevante Mutationen in trans-agierenden Faktoren des 3’ processings, sowie Beispiele für Erkrankungen oder Schäden.

Answer 29

• PAP (lost-of function):
  Cell arrest in G0-G1 phase
• PAP (Gain-of function):
  Confers high proliferative activity
  Overexpression in human carcinomas
  Hematological malignancies

Question 30

Nenne ein Beispiel, das die Wichtigkeit der Konservierung der AAUAAA-Hexanuklleotidequenz (3’-processing) zeigt

Answer 30

single-nucleotid Mutationen in der Hexanukleotidsequenz des Betaglobin gens.
Folge: Inaktivierung oder Inhibition von Globingenexpression

Question 31

Nenne drei Gain of Function Mutationen von RNA Sequenzelementen

Answer 31

• gain-of function mutation : CG -> CA:
  generation of efficient cleavage site
• gain-of function mutation : CG -> TG: generation of efficient cleavage site
• gain-of function mutation introduction of additional U-residues (20221): generation of an efficient CstF binding site

Question 32

Nenne eine mögliche Folge einer Gain of Function Mutation in RNA-Sequenzelementen

Answer 32

• raised prothrombin (coagulation factor II, F2) plasma concentration: disturbing the balance between pro- and anticoagulatory activities

increased risk to develop thrombosis

Question 33

Nenne ein Beispiel für den Effekt von alternativer poly-A-site Erkennung

Answer 33

Expression von IgM heavy chains unterscheidet sich zwischem membrangebundener und sekretierter Form.

In B-Zellen:
Low concentration of CstF-64 and high
conc. of hnRNP F or U1A protein:
CstF-64 is supressed by hnRNP or U1A
selection for M2-site - μm form is expressed

In Plasmazellen:
High concentration of CstF-64 and low
conc. of hnRNP F or U1A protein:
selection of S-site - μs form is expressed

Question 34

Nenne die drei Typen von alternativer Polyadenylierung

Answer 34

Type I: only one polyadenylation signal is present in
the 3′ UTR, thus resulting in only one mRNA isoform.

Type II: more than one resulting mRNA is produced,
but with no effect on the encoded protein (different
stability/translatability/other downstream effects)

Type III: alternative polyadenylation involves
alternative polyadenylation signals that are present
in upstream introns

Question 35

Nenne drei Faktoren, welche die Auswahl der Poly-A-site bestimmen

Answer 35

(1) Intrinsic strength of sequence elements
(2) Concentration or activity of polyadenylation factors
(3) Tissue- or stage-specific regulatory factors

Question 36

Welche Auswirkung hat Influenza auf das 3’-Processing?

Answer 36

In influenza A virus-infected cells, the highly abundant NS1 protein interacts with the cellular 30-kDa subunit of CPSF (Cleavage and Polyadenylation Specific Factor) and PABPN1 (polyadenylate-binding nuclear protein 1).

This prevents binding of the CPSF complex to its RNA substrates and selectively inhibits 3’ end processing and nuclear export of host pre-mRNAs.

The 3’ terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of 4–7 uridines in the virion RNAtemplates.

Question 37

Ist der RNA-Export mono- oder bidirektional?

Answer 37

Mono, von nukleus zu cytoplasma

Question 38

Durch welche Struktur läuft der Transport ab?

Answer 38

Kernporen

Question 39

Kann die RNA aleine transportiert werden?

Answer 39

Nein, Transportfaktoren sind nötig

Question 40

Nenne zwei Quellen für Kernexportfehler

Answer 40

pre-mRNA Mutation, folgend fehlerhafte Verarbeitung, dementsprechend keine Erkennung durch Exportmaschinerie

Mutation in Export- oder Verarbeitungsfaktoren: Trans.agierende Faktoren binden an mRNA, blockieren Export

Question 41

Nenne drei Konsequenzen von pre-mRNA Mutationen, die den Export verhindern

Answer 41

• no export of mutated transcript
• Selective downregulation of encoded protein product

-Tissue-specific dependency on expression of retained transcript

Question 42

Nenne drei Konsequenzen von
Mutation in Export- oder Verarbeitungsfaktoren, die den Export verhindern

Answer 42

-diminshed export of aggregated message

• Interacting trans-factors are sequestered from their normal nuclear duties

-Tissue-specific dependency on trans-factor activity

Question 43

Nenne mindestens vier Erkrankungen, die durch Mikrosatelit-mRNA erweiterungen hervorgerufen werden

Answer 43

Fragile X-associated tremor ataxia syndrome, FXTAS;
Premature ovarian insufficiency, POI;
Huntington’s disease, HD;
spinocerebellar ataxias, SCAs;
dentatorubral pallidoluysian atrophy, DRPLA;
spinal and bulbar muscular atrophy, SBMA;
Huntington’s disease like2, HDL2

Question 44

Nenne drei Mechanismen, die zu Erkrankungen durch Mikrosatelliterweiterungen führen

Answer 44

• Loss-of-protein function
• Gain-of-aberrant protein function due to expansions of triplet repeats within the open reading frame
• Gain-of-function of the RNA containing the expansion

Question 45

Beschreibe die Ursachen von myotonischer Dystrophie

Answer 45

CUG Repeat Sequenzverlängerung von 80 auf 2500 repeats
Folgen:
- Bindung von MBNL1 (Muscleplind Protein), Dadurch Loss of protein function

-Aktivierung von Proteinkinase C -> Hyperphosphorilierung von CUGBP1 (paralogprotein zu CELF), dadurch Stabilisierung und Funktionsverstärkung von CUGBP1

-> Störung des mRNA splicings: MBNL1 verstärkt expression von adultem, CUGBP1 von fetalem Spliceschema in mehreren mRNAs (z.B. Muskel mRNA zu CLCN1; Gehirn mRNA zu GRIN1 proteinen). Folge des Expressionshifts: Reversion zu embryonalem Splicemuster abhängig von stärke des shifts.

Folgen u.a.: Insulinresistenz, Myotonie, Kreislaufstillstandsrisiko

Question 46

Nenne eine Behandlungsmethode, sowie die Wirkweise, für Spinale Muskelatrophie auf mRNA-Ebene, welche, seti 2016, für klinische Anwendung zugelassen ist

Answer 46

Nusinersen

• Antisense oligonucleotides (AOs) or bifunctional AOs complementary to exon 7 and conjugated to splicing-enhancing effectors (e.g., serine-arginine (SR) peptide or an ESE that recruits SR proteins) are used to promote exon 7 inclusion of the SMN2 gene
• decreasing the production of a truncated SMN protein (SMND7) and increasing that of a full-length functional protein (SMN)

Question 47

Nenne zwei weitere therapeutische Ansätze, die entweder (pre-)mRNA nutzen und/oder targetieren

Answer 47

Transport von komplementären Sequenzen zu target mRNA zur Beeinflussung des splicepatterns mittels U1 oder U7 snRNA basierten Vektoren.
z.B. Duchenne: U7 antisense snRNA targetiert exon 51 (enthält Stopcodon durch Mutation). Führt zu Skipping von Exon 51 -> verkürztes, partiell funktionales Dystrophin)

RNA interferenz mittels siRNA. Elimination schädlicher mRNA.
Beispiel: Knockdown von mutierter ataxin-1 mRNA (verursacht spinocerebellare Ataxie type I (SCA1)) in Mäusen