RNA-Binding Motifs: structure, function, biological significance & human disease

1. RNA-Binding Motifs:structure, function,biological significance & human disease

2. Common RNA binding motifs

3. RNA binding motifs are the functional domains of many RNA binding proteins (RBP) • RBP are characterised by the presence of RNA bindingmotifs/domains (RBD) • RBD’s can be present in single or multiple copies • They associate with RNA in a sequence and structure dependent manner • RBP are key components in RNA metabolism: • Biogenesis: binding of RBPs and RNA molecules • Maturation: splicing, polyadenylation, capping • Transport: nuclear export • Surveillance: exon junction complex, Poly-A & cap • Translation: ribosome binding, • Editing: post-transcription & post-translational modification • Degradation: nonsense mediated decay, translation repression • Altering the expression of RBPs can have profound implications for cellular physiology • Defects in RNA metabolism caused by aberration of RBPs underlie a broad spectrum of complex human disorders

4. mRNA pathway miRNA pathway

5. RNA-recognition motif RRM RNP • RRM is by far the most common and best characterized of the RNA-binding modules. • Composed of 80–90 amino acids that form a four-stranded anti-parallel β-sheet with two a helices packed against it: (βαββαβ) • More than 10,000 RRMs have been identified • Function : • post-transcriptional gene-expression processes • ~0.5–1% of genes contain an RRM, often in multiple copies in the same polypeptide • Found predominantly in RNP • RNP, Ribonucleoprotein: a complex that contains proteins and RNA • The RNP motif refers to 2 conserved sequence elements found in the RRM • RNP1=8 amino acid conserved region at C-terminal & • RNP2= hydrophobic region toward the N-teminal • The 2 domains are located in the central b-domains and they participate in RNA recognition

6. Mutations of RRM RNA binding proteins & human disease • RRM motifs are found in a wide range of human RNA binding proteins, including: CUG-binding protein & polyA binding protein Myotonic Dystrophy: • CUG-BP is expressed at increased levels and with altered function in Myotonic dystrophy patients in a response to the CUG expansions in DMPK1 & ZNF9 • The increased levels of CUG-BP is thought to alter splicing of a number of physiological target RNA’s: • CIC-1 (muscle specific chloride channel) • IR (insulin receptor) • cTNT (cardiac troponin T)

7. Oculopharyngeal muscular dystrophy (OPMD) • Caused by poly alanine expansions in exon 1 of poly (A)-binding protein N1 (PABPN1) • PABPN1 regulates nuclear polyadenylation by binding to poly A tail of the nascent transcript with high affinity and specificity • It contains an RRM motif thought to be involved in binding to the mature mRNA • Once bound PABPN1 stimulates poly (A)-polymerase (PAP) & cleavage & polyadenylation specific factors (CPSF) • Increased poly-alanine expansion beyond a certain threshold (12-17) abrogates proper protein function • OPMD also represents a toxic RNA gof disorder whereby mutant PABPN1 accumulates in nuclear foci of skeletal muscle tissue

8. K-homology domain (KH) • KH domain so called because it was first identified in heterogeneous nuclear ribonucleoprotein-K (hn)RNP K (KH domain) • KH domains bind both ssRNA and ssDNA and is ubiquitous in eukaryotes • Composed of ~70 amino acids with a functionally important signature sequence of (I/L/V)IGXXGXX(I/L/V) near the centre of the domain. • They form a three-stranded β-sheet packed against three α-helices • Can be separated into subfamilies based on their topology • Binding & recognition is achieved by hydrogen bonding, electrostatic interactions and shape complementarity.

9. Mutations of KH RNA binding proteins & human disease Fragile X syndrome • FMR1: 2 KH domains, an RGG box, and a further RNA binding domain within the N-terminal • FMR1 encodes FRMP which belongs to the hnRNP family of proteins • Direct mutation of KH domain cause severe fragile X phenotype • Asp304Ile within 2nd KH domain causes altered RNA-binding activity • The mutated FRMP cannot: • Form homodimers • Inhibit translation • Inhibit formation of initiation complex 80S • It shuttle more rapidly between cytoplasm and nucleus

10. Nova RBPs: • Nova RBPs contain multiple KH domains and are expressed in specific regions of the brain • They regulate alternative splicing of mRNAs involved in inhibitory synaptic transmission • POMA- paraneoplastic opsoclonus-myoclonus ataxia • Auto-immune disorder that occurs when patients develop immune responses to the onconeural antigens expressed by their tumours • POMA patients show high levels of auto-antibodies generated as a result of breast, gynaecologic, lung and bladder cancers • These auto-antibodies recognize Nova RBP and abrogates its RNA binding ability • The key feature of POMA is the dysregulation of inhibitory signal to the brain and spinal motor neurons which results in the ataxia phenotype

11. Double stranded RNA binding domains (dsRBD) • ab-domain of 70-90 amino acids • 2nd most common type of RNA binding motif • Interacts with dsRNA without making specific contacts with the nucleobases • Intermolecular contacts are sequence independent and involve 2’OH group of the phosphate backbone • The presence of multiple dsRBD motifs allows recognition of particular arrangements of RNA helices which increases specificity • N-terminal helix can bind irregular helical structures e.g. hairpins

12. Mutations of dsRNA binding proteins & human disease • Adenosine deaminases which act on dsRNA’S (ADAR’s) • Involved in the most common form of RNA editing • ADENOSINE – INOSINE post-trascriptional & post-translational modification • ADAR structure = multiple dsRNA binding domains at the N-terminus + catalytic deaminase domain at the C-terminus • Majority of ADAR edited transcripts are expressed in the CNS • Glutamate-gated ion channel receptors GluRB (a component of Ca2+ AMPA channel) • GluRB undergoes physiologically fundamental editing: • Glutamine – Arginine deep within ion channel which regulates Ca2+ permeability of the AMPA receptor • Under-editing of this residue increases Ca2+ permeability with pathological consequences in the brain • Temporal lobe epilepsy • Glioblastoma multiform brain tumours

13. Autosomal dominant Dyschromatosis symmetrica is caused by mutations of ADAR1 • Mutations throughout the gene cause this syndrome with symmetrical hypo & hyper pigmentation • Toll-like receptors (TLRs): • Pathogen recognition by TLRs initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity • TLR3 is activated by binding of dsRNA, of viral or other pathogen replication intermediates, via its dsRNA binding domain • When bound to these pathogen derived ligands TLR3 initiates the cytosolic signaling cascade – inflammatory response • SNPs of TLR linked to complex common phenotypes including immunodeficiency and asthma

14. Translational repression via the miRNA pathway involves proteins with dsRBDS • MicroRNAs are small noncoding 18- to 24-nt RNAs that regulate diverse cellular and molecular processes, including cell death and proliferation • Pri-mRNAs are first processed into ~70 nucleotide pre-mRNAs by Drosha inside the nucleus. • Pre-mRNAs are transported into the cytoplasm by exportin 5 and are processed into miRNA duplexes by Dicer. • Dicer also processes long dsRNA molecules into small interfering RNA (siRNA) duplexes. Only one strand of miRNA duplex or siRNA duplex is preferentially assembled into the RNA-induced silencing comlex RISC. • RISC acts on its target by translational repression or mRNA cleavage, depending on the level of complementarity between the small RNA and its target open reading frame.

15. miRNA pathway continued…. • In human cancer, miRNAs may function as either oncogenes or tumor suppressor genes. • Increasing evidence shows that expression of miRNAs is deregulated in human cancers • More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought. • Overexpressed miRNAs in cancers, such as mir-17-92, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis. • Underexpressed miRNAs in cancers, such as let-7, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis. • miRNA expression profiles may become useful biomarkers for cancer diagnostics. • miRNA therapy could be a powerful tool for cancer prevention and therapeutics.

16. Summary • RNA binding motifs exist in many diverse proteins and can bind many different RNA species • They are the major functional components of RNA binding proteins • RBPs are of huge physiological importance and defects within the RNA binding motifs or defects which inhibit RBP function have been implicated in many human diseases from inherited ataxias and neurological disorders through to cancers which is a reflection of their essential role in many cellular processes

17. The complexity and intricacies of RNA binding proteins

18. References • The dsRNA binding site of human Toll-like receptor 38792–8797 PNAS June 6, 2006 vol. 103 no. 23 • Nature Immunology5, 975 - 979 (2004) • RNA-binding proteins: modular design for efficient function. Nature Rev Molec. Cell. Biol. 2007. • RNA modifications and human disease Advances in Oncology and Edocrinology, 2007. • RNA-binding proteins in human genetic disease. Trends in Genetics vol 24, 2008 • Cell specific RNA-binding proteins in Human disease. TCM. Vol 13, no 5, 2003 • From Mrnp trafficking to spine morphogenesis: the roots of fragile X syndrome. Nature reviews, neuroscience. May 2005 • microRNAs as oncogenes and tumor suppressors. Dev. Biol. 2007