mRNA biogenesis and disease Flashcards
Eukaryotic mRNA requires several steps processing steps in the nucleus. Mention the steps.
RNA Pol II (transcription) -> Primary transcript -> Cap -> Pre-mRNA splicing -> 3’end processing -> Add Poly(A) -> mRNP package -> Mature mRNP (mRNA export to cytoplasm) -> mRNA translation -> folded protein
In which direction is the transcribed starnd being read?
3’ to 5’ direction
In which direction is the mRNA being transcribed?
5’ to 3’ direction
What is the importance of Pre-mRNA Splicing in mRNA biogenesis?
- Larger genetic variability
- multiple proteins from one gene (via alternative splicing)
- new genes by exchange of exons
- post-transcriptional regulation of gene expression - ncreased stability: Short exons will remain more likely intact with regards to recombination events
- Exons often coding for functional domains
How many introns and exons are found in a typical primary transcript or pre-mRNA of a human?
7-8 introns and 8-9 exons
Which parts of the exon/intron are universally conserved?
- GU and AG dinucleotides at the exon-intron and intron-exon junction
- A nucleotide in the branch point (in the intron)
What are the two catalytic steps of pre-mRNA splicing, involving two consecutive trans-esterification reactions?
- an A-residue in the branch point sequence, carries out a nucleophilic on the 5‘ splice site. The splicing intermediates are exon 1 and lariat-exon
- exon 1 attacks the 3‘ splice site to generate the splicing products - spliced exon and lariat intron
What is alternative splicing?
A process by which a single transcript yields different mature mRNAs leading to the production of protein isoforms with diverse or even antagonistic functions
What is the use of alternative splicing? How often does it occur in humans?
- Alternative splicing provides tremendous opportunities for enrichment of the transcriptome and proteome without the need for expansion of the genome
- Recent estimates in humans indicate:
- 74% of multi-exon genes are alternatively spliced
- 30% of alternative splicing events produce mRNA haboring premature stop-codons (PTCs)
Name some examples of proteins encoded by human genes, that contain a large number of exons.
- Titin (Ttn; 316 exons)
- Ryanodine receptor 1 (Ryr1; 106 exons)
- Dystrophin (DMD; 79 exons)
What are the conserved RNA sequence elements involved in alternative splicing regulation?
- branch point (BP, U2 binding site)
- 5‘ splice site (U1 binding site)
- 3‘ splice site (U2AF heterodimer binding site)
- splice sites of an alternatively spliced cassette exon
- Mutations within the sequences that disrupt these interactions are responsible for 9-10% of the genetic diseases that are caused by point mutations
Discuss the general pre-mRNA splicing mechanism.
- Pre-mRNA splicing involves the identification of intron-exon boundaries (splice sites) and two successive transesterification reactions (catalytic steps)
- The dynamics of spliceosome assembly and the exchanges of snRNPs and other factors are driven by ATP consuming RNA-helicases and result in spliceosome activation, catalysis and product release
- Key RNA-RNA interactions occur during the process middle (5’ss recognition -> BP recognition -> 5’ss & 3’ss proofreading -> 5’ss transfer -> Formation of active site -> BP positing -> Branching -> 3’ss & 5’ss docking -> Exon ligation -> Intron release)
Splicing in disease: disruption of the splicing code and the decoding machinery.
Which elements can such mutations affect?
- Splice site signals (gu, ag, branchpoint)
- Exonic splicing enhancers (ESE)
- Exonic splicing silencers (ESS)
- Intronic splicing enhancers (ISE)
- Intronic splicing silencers (ISS)
What is the first layer of the splicing code?
The first layer of the splicing code consists of consensus splice site sequences positioned at exon-intron junctions (gu, ag, branchpoint)
What is the second layer of the splicing code?
The 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
How is alternative splicing regulated?
- Regulation of alternative splicing involves a dynamic interplay between antagonistic factors: SR proteins and hnRNP proteins
- ESS or ISS: motif that inhibits or silences splicing of the pre-mRNA - often bound by hnRNP proteins
- ESE or ISE: motif that directs, or enhances, accurate splicing of pre-mRNA into mRNA - often bound by members of the SR protein family
- Disruption of this antagonism can have significant consequences resulting in disease
Which proteins are ESS and ISS often bound by?
hnRNP proteins
Which proteins are ESE and ISE often bound by?
SR protein family
What role can splicing play in diseases?
- be the direct cause of the disease
- modify the severity of the disease
- determine the disease susceptibility
What are the two main categories of diseases that can occur due to splicing?
- disruption of cis-acting sequence elements (effects in cis: impact on the expression of the same (e.g.one) gene)
- affecting trans-acting factors (effects in trans: potential to affect multiple genes)
What is menat by Gain-of-splicing-function mutation?
- If a splicing element is enhanced or created (creation of cryptic splice site, ESS, ISE and ISS elements)
- beta thalassemia: creation of cryptic 3‘splice site in the first intron
- Spinal muscular atrophy (SMA)
- Duchenne muscular dystrophy disease (DMD)
- Frontotemporal Dementia (FTDP-17)
- Cystic fibrosis (CF)
What is meant by Loss-of-splicing-function mutation?
- If a splicing element is weakened or destroyed (e.g. disruption of an ESE or ESS)
- Exon 3 skipping in familiar isolated growth hormone deficiency (IGHDII)
What is meants by Mutations and alterations of splicing factors?
- If a splicing factor for constitutive or alternative splicing is mutated or non-genetically altered
- PRPF3, PRFP8 and PRFP31 in retinitis pigmentosa
What is Spinal muscular atrophy (SMA)?
- SMA refers to a number of different disorders, all having in common a genetic cause and the manifestation of weakness due to loss of the motor neurons of the spinal cord and brainstem
- Muscle-controlling nerve cells (motor neurons) are located mostly in the spinal cord
- Motor neurons are connected to muscles in the limbs and trunk
- Signals from the neurons to the muscles cause muscles to contract
- In SMA, motor neurons are lost, and muscles can’t function
Which muscles are usually more affected in SMA?
Muscles closer to the centre (proximal muscles) are usually more affected than muscles more distal from the centre (distal muscles)
What is the cause of SMA?
- SMA is caused by homozygous loss of the survival of motor neurons 1 (SMN1) gene; SMN protein is required for the assembly of the core snRNPs in the cytoplasm
- In humans there are two SMN genes, SMN1 and SMN2, on chromosome 5 both of which encode the same protein
- Mutations of the SMN genes
What is the function of SMN proteins?
- The SMN protein is critical for the assembly of the Sm-core in the snRNP biogenesis
- Sm core assembly is directed by the SMN (survival of motor neurons) complex
- This complex is an ATP-dependent assemblyosome composed of the SMN protein, Gemins 2–8, and Unrip, which specifically identifies snRNAs and brings them together with Sm proteins
- The biogenesis of snRNPs is a highly complex process involving export of the nascent pre-U snRNAs to the cytoplasm, where assembly of Sm cores takes place and, after processing of the snRNAs, reimport of the snRNPs to the nucleus to function in splicing
What determines the severity of spinal muscular atrophy (SMA)?
- Gain-of-ESS-function mutation
- Severity of SMA is modified by the production of SMN protein encoded by the paralog SMN2
- C-to-U transition inactivates an ESE and at the same time creates a new ESS that is believed to increase exon 7 skipping and the production of a truncated protein (SMNΔ7)
- Only full-length SMN2 protein is active: therefore, the greater the skipping of exon7, the more severe are the symptoms among SMA patients
What causes Duchenne muscular dystrophy disease (DMD)?
- 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 (DMD) because the mRNA lacking exon 31 produces a partially functional protein.
What causes Frontotemporal Dementia (FTDP-17)?
- 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 Frontotemporal Dementia (FTDP-17)
- The example shown is the N279K mutation (Asparagine to Lysine substitution), which enhances an ESE function, promoting exon 10 inclusion and shifting the balance toward increased expression of the 4R-Tau protein variant
What causes cystic fibrosis (CF)?
- 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
What causes Retinitis pigmentosa?
- Retinitis pigmentosa - One of the most common forms of blindness affecting 1 in 4000 people worldwide
- Disease results from retinal degeneration due primarily to progressive loss of photoreceptor cells
- Most cases are sporadic, but mutations in more than 30 genes cause familial forms of the disease
- Three dominant retinitis pigmentosa genes (PRPF31, PRPF8, PRPF3- pre-mRNA-processing factor gene) encode proteins required for function of U4.U5.U6 tri-snRNP assembly of the spliceosome
- Cell type specificity is likely due to sensitivity of one or more photoreceptor-specific pre mRNAs to loss of U4.U5.U6 tri-snRNP function
- Normal spliceosome: Functional PRPF proteins (e.g., PRPF3, PRPF8) and snRNAs (U4/U6) enable proper splicing, maintaining normal photoreceptor cells.
- Mutant PRPF proteins: Mutations (e.g., in PRPF3, PRPF31) disrupt spliceosome assembly/activity, leading to a defective spliceosome and aberrant splicing.
- Consequence: Aberrant splicing likely triggers photoreceptor loss
What are the critical RNA recognition motifs found in mRNA for 3’ end processing?
- USE = upstream sequence element
- AAUAAA = poly(A) signal
- CA = Cleavage site
- DSE = downstream sequence element
What are the steps in 3’ end mRNA processing?
- Assembly of multiprotein complexes at specific RNA recognition motifs
Several protein complexes are involved in 3’ end processing:
- CPSF= cleavage/ polyadenylation specificity factor
- CstF= cleavage stimulating factor
- CF I= Cleavage factor I
- CF II= Cleavage factor II
- PAP= poly(A) polymerase - Cleavage at the cleavage site by CPSF 73
- Formation of a poly(A) tail by PAP (addition of ~250 A residues) that is bound by PABP
Are cis- or trans-acting elements involved in 3’end processing?
Cis-acting sequence elements and trans acting factors are involved in mammalian 3’ end processing
What are the two main categories of errors in 3’end processing?
- Mutations of sequence elements (DSE, AAUAAA, CA, USE)
- Role for trans-acting factors
What are types of Mutations of sequence elements (DSE, AAUAAA, CA, USE) of 3’end processing and examples of their corresponding diseases?
- Loss-of-function mutation (AAUAAA): Alpha thalassemia (alpha-globin gene), Beta thalassemia (beta-globin gene), IPEX syndrome (Fox3p gene), Fabry disease (alpha Gal A gene, AAUAAA signal within ORF, no 3’UTR)
- Gain of function mutation (CA): Thrombophilia – prothrombin, Thrombophilia – fibrinogen
- USE - important for 3’ processing: Lamin B, Complement C2, Cyclooxygenase 2 (Cox 2), Collagen
- Alternative poly(A) signal recognition: IgM heavy chain pre-mRNA during B-cell differentiation
What are the types of errors due to trans-acting factors of 3’end processing and their corresponding effects?
- PAP (loss-of-function): Cell arrest in G0-G1 phase
- PAP (Gain-of-function): Confers high proliferative activity, Overexpression in human carcinomas, Haematological malignancies
What causes thalassemia?
- Mutations of sequence elements loss-of-function mutations can cause thalassemia
- Mutations that alter the AAUAAA hexanucleotide sequence element
- Inactivate or inhibit globin gene expression
- These mutations show functional importance of the highly conserved poly(A) signal with very little sequence flexibility
What could cause thrombosis?
- gain-of-function mutation at CG -> CA: generation of efficient cleavage site
- gain-of-function mutation at CG -> TG: generation of efficient cleavage site
- gain-of-function mutation introduction of additional U-residues (20221): generation of an efficient CstF binding site
- Raised prothrombin (coagulation factor II, F2) plasma concentration: disturbing the balance between pro- and anticoagulatory activities -> increased risk to develop thrombosis
Give an example of a system, where alternative 3‘end processing affects B-cell differentiation.
- B-cell differentiation: switch of IgM heavy-chain expression from a membrane-bound form (µm) to the secreted form (µs)
- CstF-64 binds with different affinities to the IgM transcript binding sites (S: low affinity; M2: high affinity)
- Low concentration of CstF-64 and high concentration of hnRNP F or U1A protein: selection of M2-site -> µm form is expressed
- High concentration of CstF-64 and low concentration of hnRNP F or U1A protein: selection of S-site -> µs form is expressed
What are the Classifications of alternative polyadenylation?
- 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
Are cis- or trans-acting elements involved in the regulation of alternative polyadenalation?
Alternative poly (A) site choice: regulated by various cis- and trans-acting determinants:
- Intrinsic strength of sequence elements
- Concentration or activity of polyadenylation factors
- Tissue- or stage-specific regulatory factors
What do regulated and alternative 3’ end processing modulate?
Regulated and alternative 3’ end processing modulates the temporal and spatial diversity of gene expression
Discuss Influenza A virus and its connection to the cellular 3’ end apparatus.
- In influenza A virus-infected cells, the highly abundant NS1 protein interacts with the cellular 30-kDa subunit of CPSF and PABPN1.
- This prevents binding of the CPSF complex to its RNA substrates and selectively inhibits 3’ end processing and nuclear export of host pre-mRNAs.
- This allows the virus to suppress host gene expression, including the expression of antiviral genes, thereby facilitating viral replication and evasion of the host immune response.
- 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 RNA-templates.
- This mechanism ensures that viral mRNAs are properly polyadenylated and can be exported from the nucleus and translated, even though host mRNA processing is inhibited.
What are examples of transport receptors and the type of RNAs they transport?
- Crm1 for HIV RNA, U snRNA, rRNA, 5S rRNA, mRNA
- Exp-t for tRNA
- Exp-5 for miRNA
What are the two sources of mRNA nuclear export defects?
- Mutations in pre-mRNA sequences
- Mutations in export or processing factors
What is meant by Mutations in pre-mRNA sequences and how does it affect the nuclear export?
- Mutation in a particular mRNA can cause improper processing and inability to be recognized by the export machinery (Osteogenesis imperfecta type I)
- No export of mutated transcript
- Selective downregulation of encoded protein product
- Tissue-specific dependency on expression of retained transcript
What is meant by Mutations in export or processing factors and how does it affect the nuclear export?
- Abnormal sequence expansion in an mRNA can result in nuclear retention and sequestering of trans-acting factors (Myotonic dystrophy type I (DM1))
- Diminished export of aggregated message
- Interacting trans-factors are sequestered from their normal nuclear duties
- Tissue-specific dependency on trans-factor activity
What can Microsatellite expansion in RNA cause?
Nuclear retention and disease
What are the three mechanisms by which microsatellite expansions can cause diseases?
- 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
What are some examples of diseases cause by microsatellite expansions within transcribed regions with confirmed or potential RNA gain-of-function effects?
- Fragile X-associated tremor ataxia syndrome, FXTAS – 5’UTR
- Premature ovarian insufficiency, POI – 5’UTR
- Huntington’s disease, HD - ORF
- Spinocerebellar ataxias, SCAs – ORF, SCA12 – 5’UTR, SCA10 – Intron
- Dentatorubral pallidoluysian atrophy, DRPLA - ORF
- Spinal and bulbar muscular atrophy, SBMA - ORF
- Huntington’s disease like2, HDL2 – 3’UTR
What is an example of a disruption of alternative splicing by an RNA Loss-of-Function?
- Sequestration of muscleblind protein MBNL1 at mutated DMPK mRNAs in nuclear foci in myotonic dystrophy (DM1) patients
- Myotonic Dystrophy, DM2 – Intron, DM1 – (CUG)n at 3’UTR
What is an example of a disruption of alternative splicing by an RNA Gain-of-function?
- Activation of protein kinase C (PKC) by the expanded CUG RNA induces hyperphosphorylation of CUGBP1(paralogue protein CELF). This modification stabilizes the protein.
- CUGBP 1 – (CUG)n at 3’UTR
What do expanded CUG repeats in DM1 result in?
- MBNL1 loss-of-function and in CUGBP1 gain-of-function
- The levels of MBNL1 and CUGBP1 in the nucleus control a subset of developmentally regulated splicing events that are reversed in DM1
- Upregulation of CUGBP1: DM1 leads to an abnormal increase in CUGBP1 levels -> leads to insulin resistance, myotonia (CIC-1), and possibly cardiac defects (cTNT)
What therapeutic approach can be used against SMA?
- 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)
- antisense oligonucleotide known as nusinersen (approved for clinical use in 2016)
What therapeutic approach can be used against DMD?
- Modified U1 or U7 snRNA-based vectors can carry complementary sequences to the targeting RNAs to achieve the desired splicing pattern
- Genomic deletions (e.g., from exon 48 to 50) in dystrophin genes of DMD patients lead to a premature termination codon (STOP) in exon 51 caused by a frameshift
- Antisense U7 snRNA targeting the 3‘splice site of exon 51 prevents the inclusion of this exon and restores the reading frame and, as a result, produces a partially functional dystrophin protein
- Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy
What therapeutic approach can be used against SCA1?
- RNAi approaches are used to eliminate pathogenic mRNAs.
- siRNAs delivered to a mouse model of spinocerebellar ataxia type I (SCA1) elicit degradation and efficient knockdown of the disease causing ataxin-1 mRNA that contains the expanded CAG repeats (in ORF) encoding polyglutamine (polyQ).
What therapeutic approaches are used to target pre-mRNAs or mRNAs in cis-acting splicing defects?
Gene specific (ASOs, CRISPR)
