Recomendados
Más contenido relacionado
La actualidad más candente
La actualidad más candente (20)
Destacado
Similar a miRNA & siRNA
Similar a miRNA & siRNA (20)
Último
Último (20)
miRNA & siRNA
- 1. Origins and Mechanisms of miRNAs and siRNAs Present by: Mozhdeh Mirahadi 1
- 2. RNAi Overview During RNAi Double-stranded RNAs cut into short double-stranded RNAs, s(small) i(interfering) RNA's, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi 2
- 3. Outlines • IInnttrroodduuccttiioonn • RNA silencing • Definition of RNA interference • MMeecchhaanniissmm ooff RRNNAA iinntteerrffeerreennccee • Argonaute • siRNAs; Sources of siRNA Precursors • RISC • Posttranscriptional Silencing by siRNAs • MicroRNAs • MicroRNA Biogenesis • Posttranscriptional Repression by miRNAs • CCoonncclluussiioonn 3
- 4. Introduction RNA i ( RNA Interference ) 4
- 5. Definition RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons. 5
- 6. Need for interference Defense Mechanism Defense against Infection by viruses, etc As a defense mechanism to protect against transposons and other insertional elements Genome Wide Regulation RNAi plays a role in regulating development and genome maintenance. 30% of human genome regulated 6
- 7. RNAi: Silencing in Cenorhabditis elegans dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function. 7
- 8. Cenorhabditis elegans RNAi can be induced in C. elegans in three simple ways: Injection of dsRNA into the worm gonads Soaking the worms in dsRNA solution Feeding the worms engineered bacteria producing dsRNA 8
- 9. Mechanism of RNAi RNA i ( RNA Interference ) 9
- 10. 10
- 11. The Players In Interference RNA siRNA: dsRNA 21-22 nt. miRNA: ssRNA 19-25nt. Encoded by non protein coding genome RISC: RNA induced Silencing Complex, that cleaves mRNA Enzymes Dicer : produces 20-21 nt cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre miRNA; which is later processed by Dicer 11
- 12. siRNAs • Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing • RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation • A single base pair difference between the siRNA template and the target mRNA is enough to block the process. • Each strand of siRNA has: • a. 5’-phosphate termini • b. 3’-hydroxyl termini • c. 2/3-nucleotide 3’ overhangs 12
- 13. siRNA design 21-23nt 2-nt 3' overhangs ( UU overhangs ) G/C content: 30-50%. No basepair mismatch Synthesised siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). BLAST : eliminate any target sequences with significant homology to other coding sequences. 13
- 14. Generation of small interference RNA 14
- 15. miRNA Originate from capped & polyadenylated full length precursors (pri-miRNA) Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA) 15
- 16. Difference between miRNA and siRNA Function of both species is regulation of gene expression. Difference is in where they originate. siRNA originates with dsRNA. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. miRNA originates with ssRNA that forms a hairpin secondary structure. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target. And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre-transcriptionally (RNA induced transcriptional silencing or RRIITTSS) 16
- 17. Dicer RNase III-like dsRNA-specific ribonuclease Enzyme involved in the initiation of RNAi. It is able to digest dsRNA into uniformly sized small RNAs (siRNA) Dicer family proteins are ATP-dependent nucleases. Rnase III enzyme acts as a dimer Loss of dicer→loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer is a conserved protein) 17
- 18. RISC RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA Unwinding of double-stranded siRNA by ATP independent helicase. The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand. 18
- 19. Summary of Players Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) Drosha and Dicer are RNase III enzymes Pasha is a dsRNA binding protein Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran and GTP Argonautes are RNase H enzymes 19
- 20. Mechanism of RNA interference 20
- 21. Mechanism of RNA interference 21
- 22. Over Figure 1. Core Features ooff mmiiRRNNAA aanndd ssiiRRNNAA SSiilleenncciinngg 22
- 23. Argonaute: At the Core of RNA Silencing The Argonaute superfamily can be divided into three separate subgroups: the Piwi clade that binds piRNAs, the Ago clade that associates with miRNAs and siRNAs, third clade that has only been described thus far in nematodes. 23
- 24. Over Figure 2. A Diversity ooff ssiiRRNNAA SSoouurrcceess 24
- 25. RISC Assembly and siRNA Strand Selection Although single-stranded siRNAs can load directly into purified Argonaute proteins, the double-stranded siRNAs that are generated by Dicer cannot and rely instead upon siRISC assembly pathways (Figure 2). 25
- 26. Over Figure 3. Mechanisms of siRNA Silencing 26
- 27. Amplification of siRNA 27
- 28. siRNAs Can Induce Heterochromatin Formation siRNAs are not restricted to posttranscriptional modes of repression. In 2002, siRNAs were shown to induce heterochromatin formation in S. pombe, consistent with earlier reports of transcriptional gene silencing (TGS) in plants. 28
- 29. Illustration of miRNA processing 29
- 30. Another View 30
- 31. MicroRNA Biogenesis MicroRNAs in the plant and animal (Figure 4) 31
- 32. Over Figure 4. Biogenesis of miRNAs and Assembly into miRISC in Plants and Animals 32
- 33. MicroRNA Associations miRNA strand miRNA* strand In Drosophila in humans, C. elegans, and Drosophila indicates 33
- 34. Posttranscriptional Repression by miRNAs The miRNA acts as an adaptor (Figure 5) The degree of miRNA-mRNA complementarity has been considered a key determinant of the regulatory mechanism. 34
- 35. Over Figure 5. Possible Mechanisms of miRISC-Mediated Repression 35
- 36. Conclusions dsRNA needs to be directed against an exon, not an intron in order to be effective Homology of the dsRNA and the target gene/mRNA is required Targeted mRNA is lost (degraded) after RNAi The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism) ssRNA does not work as well as dsRNA 36
- 37. Thank you! Any guestion? 37
Notas del editor
- Biochemistry of RNA interference Numerous studies have investigated the biochemical mechanisms that underpin RNAi induced gene silencing (Tabara et al., 1999; Mourrain et al., 2000; Sijen et al., 2001). These studies have revealed that RNAi suppresses gene function by promoting degradation of specific mRNA involving highly specific and complex protein–protein interactions that occur in the RNA-induced silencing complex (RISC). Depending on the thermodynamic stability of the 5′-end, both the sense and antisense regions of a given siRNA can enter the RISC complex. However, the antisense strand of the siRNA, which is complementary to the target mRNA, serves to accurately identify the target mRNA and induces sequence-specific degradation in association with other components of RISC at the relatively thermodynamically unstable 5′-end. A key component of RISC is the protein argonaute-2 that binds to a single strand of siRNA. Argonaute-2 and the 5′ strand of the siRNA mediate the recognition of the target mRNA and, with other components of RISC, induce mRNA cleavage with consecutive suppression of protein translation
- http://www.ambion.com/techlib/misc/siRNA_finder.html Target prediction The secondary structure of mRNA not only influences the maturation of pre-mRNA and the translation into protein (de Smit&van Duin, 1990; Balvay et al., 1993), it also determines the efficacy of a complimentary siRNA to access its mRNA target (Holen et al., 2002; Kretschmer-Kazemi Far & Sczakiel, 2003). Notably Heale and collegues have developed a secondary structure prediction model to identify nonaccessible mRNA sites for RNAi (Heale et al., 2005). For effective gene silencing engineering of 21-nt doublestranded siRNA with a 2-nt deoxythymideine (Ts) overhang at the 3′-end has been recommended by several groups (Chiu & Rana, 2002; Elbashir et al., 2002; Paddison et al., 2002; Khvorova et al., 2003; Reynolds et al., 2004; Ui-Tei et al., 2004), because a 3′-end overhang is more efficient in guiding dsRNA to unwind. Generally synthesised siRNA should not target introns, the 5′- and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). Furthermore, the guanine (G)–cytosine (C) content of the designed siRNA should be between 30% and 50% and the 5′-ends of antisense and sense strand should have high and low thermodynamic stability, respectively. Investigators should avoid internal repeats and palindromes of siRNA. At certain positions in the sense strand of the 21-nt siRNA, base preferences may be considered: an adenosine (A) at positions 3 and 19; absence of G or C at position 19; and a uracil (U) at position 10; and absence of G at position 13. Indeed, thermodynamic properties of siRNA are critical in determining its stability and gene silencing efficacy (Khvorova et al., 2003). Finally, a BLASTsearch of the appropriate genome database should be performed and low-stringency sequences should be avoided to ensure that no other unrelated genes are targeted to minimize off-target effects. Many effective and specific siRNA have been published already and can be found in the public domain.
- Generation of siRNA for silencing of gene expression. (A) From top to below, chemically synthesized siRNA, long dsRNA that can be cleaved by Dicer to form siRNA, and shRNA that can be cleaved by Dicer to form siRNA. (B) From top to below, sense and antisense strands are expressed by RNA polymerase III promoter (e.g., U6 promoter) separately and form a double-stranded siRNA molecule, shRNA are expressed by RNA polymerase III promoter (e.g., U6 promoter) first and then cleaved by Dicer to form mature siRNA. Chemically synthesized siRNA, shRNA, and long dsRNA have been used to generate siRNA by introducing these molecules into cells. After entry into the cytoplasm, shRNA and long dsRNA are cleaved into 21-nt long mature siRNA by a RNase III (Dicer), which is an end-recognition endonuclease). These methods generally result in temporary silencing effects. However, long dsRNA can also elicit responses of the innate immune system such as interferon (IFN) release. To obtain stable and inducible RNAi, researchers have recently developed shRNA structures driven by U6 or H1 promoters (RNase III promoters), wherein the shRNA has 2 short duplex stems: one stem connected to a loop sequence, and the other ending with 6 or more thymidines (T) as the termination signal.
- miRNA (micro-RNA)http://en.wikipedia.org/wiki/MiRNA A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long. It is thought to regulate the expression of other genes. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA). In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
- Double-stranded RNA triggers processed into siRNAs by enzyme RNAseIII family, specifically the Dicer family Processive enzyme - no larger intermediates. Dicer family proteins are ATP-dependent nucleases. These proteins contain an amino-terminal helicase domain, dual RNAseIII domains in the carboxy- terminal segment, and dsRNA-binding motifs They can also contain a PAZ domain, which is thought to be important for protein-protein interaction. Dicer homologs exist in many organisms including C. elegans, Drosphila, yeast and humans Loss of dicer: loss of silencing, processing in vitro Developmental consequence in Drosophila and C. elegans Dicer is a conserved protein
- The initiation pathway may be amplified by the cell through the synthesis of a population of secondary siRNAs using the dicer-produced initiating or primary siRNAs as templates. These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).