Nonsense mediated mRNA decay Protein folding and misfolding

Nonsense mediated mRNA decay

Protein folding and misfolding

• Protein terminating codons (PTCs) are common, accounting for a third of mutations in inherited genetic disorders.

• PTCs arise when a single base change in genomic DNA directly introduces a stop codon or indirectly from a shift in the translation reading frame that is caused by a exon-skipping mutation or a genomic insertion/deletion.

• PTCs can also arise from aberrant alternative splicing events that produce a PTC-containing mRNA.

Protein terminating codons (PTCs)

The process of eukaryotic gene expression involves a number of interlinked steps - transcription, splicing, polyadenylation, capping, translation, and mRNA degradation.

• Intron splicing of pre-mRNA results in the deposition of the exon-junction complex (EJC).

• In cytoplasm, mRNA undergoes a pioneer round of translation which removes many of the proteins bound to the mRNA in the nucleus.

Wiley Interdisciplinary Reviews – RNA,Vol. 1, Is. 1, 2010

Nonsense mediated mRNA decay (NMD)

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1757-7012

• PTCs are recognized by specific factors and induces mRNA degradation – nonsense mediated mRNA decay.

• NMD is mechanism preventing the production of potentially deleterious C-terminally truncated proteins translated from mRNA containing PTCs.

• NMD plays an important role as a modulator of clinical manifestations of many genetic diseases.

• NMD can be beneficial by preventing the production of C-terminally truncated proteins with a dominant negative function.

• There are also cases where the truncated protein encoded by the PTC-containing mRNA still has some residual function and the NMD-mediated reduction of mRNA abundance results in more severe clinical problems.

• A better understanding of the molecular mechanism of NMD will be associated with the development of therapeutic approaches aiming at specifically manipulating NMD efficacy and specificity.


The mRNA transcripts are shown in a closed-loop conformation. The translation start site and normal termination (stop) site are indicated and the broken arrows indicate ribosome movement during translation.The faux 3´UTR model – the distance between the terminating ribosome and the poly(A) tail denotes the distinction between a normal codon and PTC. - Normal translation termination is characterized by the interaction of eRF3

(eukaryotic release factor) with PABPC1 (polyA-binding protein).- Premature termination prevents the eRF3–PABPC1 interaction by artificially

increasing the length of the 3´UTR and the physical distance between the two proteins.

Models of nonsense mediated mRNA decay

• The EJC is a multi-protein complex that is deposited onto mRNA 20–24 nucleotides upstream of exon–exon junctions during splicing; a recent study indicates that EJCs are deposited upstream of 80% of exon–exon junctions in human mRNAs.

• Newly synthesized mRNAs are bound at the 5´ end by the cap-binding complex (CBC), and during a ‘pioneer round’ of translation the translating ribosome scans the entire length of the CBC-bound mRNA, displacing all EJCs from the transcript until it reaches the normal translation termination codon.

• In the event that a ribosome terminates translation prematurely due to the presence of a PTC, EJCs downstream of the PTC will remain and recruit NMD effectors, forming functional NMD complexes.

Exon junction complex (EJC)

The EJC dependent model – differentiation between a normal codon and PTC requires splicing-dependent deposition of the EJC and a pioneer round of translation. - In normal translation termination, the translating ribosome traverses the entire

length of the mRNA during the pioneer round of translation and displaces all the EJCs. When it reaches the normal stop codon in the last exon, all EJCs have been displaced and the communication of eRF3 with PABPC1 signals normal termination.

- Premature translation termination is characterized by the presence of a core EJC downstream of PTC. This remaining EJC recruits the NMD effectors UPF2 and UPF3 and the SURF protein complex (SMG1, UPF1, and eRF1 and eRF3), and this EJC–SURF interaction leads to phosphorylation of UPF1 by SMG1, which initiates degradation of the PTC-containing transcript.


The ribosome release model – early ribosome release caused by the presence of a PTC exposes the downstream unprotected mRNA to degradation by nucleases. Translationally competent ribosomes normally protect the mRNA from degradation as they traverse the transcript, but at a PTC it is proposed that the interaction of UPF1 with eIF3 stimulates ribosome release. This model incorporates data indicating that phosphorylated UPF1 interacts with eIF3 to stimulate ribosome release from the mutant mRNA.


In mammals, newly synthesized CPB80–CBP20-bound mRNA is targeted for NMD once mRNA has been generated by pre-mRNA processing and exported from the nucleus to the cytoplasm. During pre-mRNA processing, splicing results in the deposition of an EJC of proteins upstream of mRNA exon–exon junctions. EJC components include eIF4AIII, Y14, MAGOH, BTZ and many other proteins. The UPF3 (UPF3a) or UPF3X (UPF3b) join EJCs in the nucleus so as to be exported with mRNA to the cytoplasm. In the cytoplasm, UPF3 or UPF3X recruits UPF2. The translation of CBP80–CBP20-bound mRNA constitutes the pioneer round. Translation termination during the pioneer round at a PTC that is situated 50–55 nt upstream of an exon–exon junction (i.e. 25–30 nt upstream of an EJC) involves the SURF complex, which consists of the PI3K-related protein kinase that phosphorylates UPF1, SMG1, together with UPF1, eRF1 and eRF3. As a consequence, NMD generally occurs. During the process, UPF1 together with SMG1 is thought to bind EJC-associated UPF2in a way that is promoted by CBP80. UPF1 binding to the EJC results in UPF1 phosphorylation. Phospho-UPF1 triggers NMD by promoting translational repression of the NMD target. Translational repression involves the binding of phospho-UPF1 to eIF3 within the 43S pre-initiation complex that is poised at the AUG translation initiation codon so as to prevent 60S ribosomal subunit joining. Phospho-UPF1 also promotes NMD by recruiting mRNA degradative activities. Not shown are SMG5, SMG6 and SMG7, which activate UPF1 dephosphorylation and thus recycling. SMG6 appears to additionally function as an endonuclease. Very recently, roles for SMG8 and SMG9 as SMG1-interacting proteins have been defined. Nucleolytic activities are indicated by the red irregular hexagons. PABP, poly(A)-binding protein, where darker shapes specify the largely nuclear PABPN1 and lighter shapes denote the largely cytoplasmic PABPC1; AUG, translation initiation codon; STOP, normal termination codon; 1, eRF1; 3, eRF3.

Maquat LE, 2010

1468C>T, R490W550delA, T184RfsX364

598-612del, F200_L204del550delA, T184RfsX363

2314-2317del,

D772delK773NfsX3245C>T, P82L2

550delA, T184RfsX36245C>T, P82L1

MUTACE DETEKOVANÁ NA ALELE 2MUTACE DETEKOVANÁ NA ALELE 1PACIENT

mRNA: homozygotní výskyt missense mutace nebo in-frame delece

DNA: heterozygotní výskyt missense mutace nebo in-frame delece + detekce frame-shift delece

® degradace mRNA nesoucí frame-shift deleci mechanismem nonsense mediated mRNA decay

Detekce mutací v genu CAPN3 (LGMD2A)


0

0,2

0,4

0,6

0,8

1

1,2

1 2 3 4 5 6 7 8 9 10 11 12

Stanovení relativního množství mRNA genu CAPN3

Relativní množství mRNA v závislosti na typu mutace:

non-PTC/non-PTC: 0,97

non-PTC/PTC: 0,37

PTC/PTC: 0,02

Pacient 1-4: non-PTC/non-PTC

Pacient 5-9: non-PTC/PTC

Pacient 10-12: PTC/PTC



• NMD zhoršuje klinické projevy nemocí – Duchennova svalová dystrofie

• NMD zmírňuje klinické projevy nemocí – Osteogenesis imperfecta

Osteogenesis imperfecta:Mutace kolagenu typu I (geny COL1A1 a COL1A2); kolagen typu I - hlavní strukturní protein kostí, mutace mají za následek náchylnost k lomivosti kostí a deformitám skeletu

http://www.cdaarthritis.com/images_slides/images_slides_11d_larger_slides.htm

• Missense mutace asociované s genem COL1A jsou příklady dominantně-negativních alel ......... ruší konformaci kolagenových podjednotek a jsou spojeny s těžkými klinickými fenotypy osteogenesis imperfecta typu II–IV.

• PTC mutace vyvolávající NMD (nevzniká mutantní protein účastnící se struktury) a jsou spojeny s mírnějšími klinickými fenotypy osteogenesis imperfecta typu I.

• Kolagen typu I – tvořen ze dvou řetězců prokolagenu α1 (COL1A1) a jednoho prokolagenu α2 (COL1A2).

Therapies based on translational read-through

PTC124 - a new drug in development for mutation-specific treatment of inherited diseases such as DMD and CF. PTC124 is able to bind the decoding centre of the ribosome and decrease the accuracy of codon–anticodon pairing. The recognition of PTC is suppressed and, instead of chain termination, an amino acid is incorporated into the polypeptide chain.PTC124 promotes read-through of PTCs without affecting normal stop codons. PTC124 is being investigated in clinical studies in CF and DMD patients.



Therapies based on translational read-throughAminoglycosides are able to bind the decoding centre of the ribosome and decrease the accuracy of codon–anticodon pairing. The recognition of stop codons is suppressed and, instead of chain termination, an amino acid is incorporated into the polypeptide chain .After successful trials in animal models, clinical trials in patients with CF or DMD were performed with gentamicin. These trials showed that aminoglycosides can promote in vivo read-through of nonsense mutations and can lead to the expression of full-length proteins. Requirement of high concentrations for a prolonged effect, the intravenous administration mode and known side effects, such as kidney damage and hearing loss, limited the usefulness of its systemic application.

Protein expression is the multistep process involving regulation at the level of transcription, mRNA turnover, protein translation, and post-translational modifications leading to the formation of a stable product.

Protein expression

Scheme of the free-energy surface that proteins explore as move towards the native state.

The accumulation of conformations that need to traverse energy barriers to reach a favourable downhill path.

When molecules fold in the same compartment, the free-energy surface of folding may overlap with intermolecular aggregation. This state is prevented by molecular chaperones.

Protein folding

NATURE | VOL 475 | 21 JULY 2011

Partially folded states are problematic - they tend to aggregate in concentration-dependent manner. Aggregation primarily results in amorphous structures. Alternatively, fibrillar aggregates called amyloid may form. Formation of these aggregates in vivo is strongly restricted by the chaperone machinery.

Molecular chaperone interact with, stabilize or help proteins to acquire its functionally active conformation, without being present in its final structure. Different classes of structurally unrelated chaperones exist in cells.

Protein folding

• The nascent proteins are synthesized vectorially and the N-terminal sequences will be available for folding before the C-terminal fragments. • Translation is coupled to co-translational folding. The rate of translation is non-uniform along mRNAs and is shaped by the asymmetric tRNA abundance for the different codons. Codons that pair to lowly abundant tRNAs are translated slower that codons read by highly abundant tRNA.• Slow-translating segments are not randomly distributed along the coding mRNA sequences; they are predominantly located downstream of the domain boundaries of multidomain proteins.

Protein folding

• HPA patients may benefit from pharmacological doses of the BH4.

• BH4 reduced blood phenylalanine concentrations and increased dietary phenylalanine tolerance by increasing in vivo PAH activity.

• In 2007, sapropterin dihydrochloride (BH4) was approved as the drug to treat patients with BH4-responsive HPA.

• PKU - protein misfolding disease• BH4 - pharmacological chaperone• A pharmacological chaperone is a small molecule that rescues protein function by improving protein folding and by stabilizing the protein structure.

Hyperphenylalaninemia (HPA), phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH4)

Molecular mode of action of BH4. At the protein level, BH4 prevents misfolding, aggregation, and degradation, and thus induces an increase in the effective PAH concentration resulting in rescue of its function. J Inherit Metab Dis (2010) 33:649–658

Hyperphenylalaninemia, tetrahydrobiopterin therapy

• Mutations in the LDLR (low-density lipoprotein receptor) or the APOB (apolipoprotein B) genes

• Autosomal dominant inheritance• Incidence: 1:500

Familial hypercholesterolemia (FH)

• LDLR is synthesized by ribosomes bound to the endoplasmic reticulum (ER), partially glycosylated (molecular mass 120 kDa).

• LDLR is transported to the Golgi apparatus, glycosylated (molecular mass 160 kDa).

• LDLR is transported to the cell surface, where it mediates the uptake of lipoprotein particles, mainly low-density lipoproteins (LDLs), by receptor-mediated endocytosis.

• The internalized LDL particle is subsequently released in the endosome, and the receptor returns to the cell surface in a process called receptor recycling.

Cell. Mol. Life Sci. Vol. 61, 2004

Familial hypercholesterolemia

• Folding of LDLR occurs in a vectorial manner, domain by domain.• The newly synthesized LDLR chains fold rapidly into compact structures containing non-native disulfide bonds linking distant regions of the protein.• With time, non-native disulfides are reshuffled, allowing extension of the molecule. In the native conformation, disulfide bonds only exist between cysteine residues within individual repeats. Despite the extensive formation of non-native disulfide bonds during folding, LDLR rarely aggregates - non-native disulfide bond formation are part of the normal LDLR folding pathway.• Efficient folding of LDLR may be caused by assistance of chaperones and folding enzymes in ER.

Familial hypercholesterolemia, protein folding

Cell. Mol. Life Sci. 61 (2004)

A molecular model of the LDLR beta-propeller. Left: the six blades in the beta-propeller. Right: side viev. The amino acids G528, G544 and W556 are shown in red.

FEBS Journal 274 (2007) 1881–1893: Transport defective mutations (class 2) causing partial or complete retention of LDLR in the endoplasmic reticulum (G544V, G528D and W556R, the beta-propeller), to study the ability of chemical chaperones to assist folding and to facilitate the transport of the mutant LDLR out of the ER.


Western blot analysis of the rescue of LDLR mutants. Stably transfected CHO cells expressing G544V-mutant, G528D-mutant or W556R-mutant LDLR were incubated with different chemical chaperones. Cell lysates were prepared, and equal amounts of proteins were subjected to SDS ⁄ PAGE and western blot analysis using an antibody to LDLR. A cell lysate from CHO cells stably transfected with wild-type (WT) LDLR is included on the left. FEBS Journal 274 (2007) 1881–1893

The wild-type LDLR appeared primarily with molecular mass of 160 kDa on the western blot, representing the mature form of the LDLR, whereas the mutant LDLRs appeared with molecular mass of 120 kDa, representing the ER-localized form of the receptor.


Confocal laser microscopy on CHO cells stably transfected with wild-type LDLR or G544V-mutant LDLR. The upper panels show cells expressing wild type LDLR, the middle panels show cells expressing G544-mutant LDLR, and the lower panels show cells expressing G544V-mutant LDLR treated with 5 mM 4-PBA for 24 h.FEBS Journal 274 (2007) 1881–1893

The appearance of a 160 kDa mature form of LDLR indicates that the receptor has escaped from the ER and has been subjected to oligosaccharide modifications in the Golgi apparatus. To determine whether the mature form of G544V-mutant LDLR appeared at the cell surface, confocal laser microscopy on intact cells was performed. The wild-type LDLR was present on the plasma membrane, whereas the G544V-mutant LDLR was almost undetectable. When the cells were grown in the presence of 4-PBA, the G544V-mutant LDLR could be observed.


• ER contains stringent quality-control systems, which ensure that only correctly folded proteins are transported to their final destinations.

• The ER quality control system involves molecular chaperones that transiently associate with newly synthesized proteins and promote their folding.

• Misfolded proteins are retained and subsequently degraded by the ER-associated degradation.

• Protein misfolding is the cause of several genetic diseases. • Chemical chaperones are small molecules that bind to a protein,

stabilize the folded state, and thereby reduce protein misfolding. It has been proposed that chemical chaperones promote folding of mutant proteins, allowing them to escape from ER retention and subsequent degradation.

• Accumulation of misfolded proteins in the ER has been shown to cause ER stress and activation of a protective response known as the unfolded protein response (UPR).

Protein folding

Documents

Nonsense mediated mRNA decay Protein folding and misfolding