Structural and functional modules in RNA interference

Abstract

RNA interference (RNAi) uses small RNA molecules to regulate transcriptional and post-transcriptional gene expression. In recent years, a number of structural studies provided insights into the molecular architecture and mechanism of functional modules of RNAi. Mechanisms of nucleic acid recognition and cleavage have been revealed by structural studies of proteins and their nucleic acid complexes involved in RNA biogenesis, for example, Argonaute, PIWI, RNase III, Dicer, Drosha, and DGCR8. While quite a few questions remain, an excellent structural and mechanistic overview of RNAi processes has already emerged. In this review, we examine functional modules and their assemblies in RNAi processes.

Figure 1. Major RNA interference pathways and domain composition of proteins involved in them

(A) Processing of RNAs in miRNA and siRNA. Pri-miRNA transcripts are processed by Drosha/DGCR8 in the nucleus and Dicer in the cytoplasm. The cleavage sites are indicated by orange triangles. siRNAs start as dsRNA and are processed by Dicer. In both cases the end product 20–30 nt RNA is incorporated into the RISC complex. (B) Model of piRNA pathway. Transposon mRNA is cleaved by PIWI using a piRNA guide strand. A fragment of cleaved transposon RNA is incorporated into Ago3 RISC and used to cleave the piRNA cluster transcript. Its fragment is incorporated into PIWI protein to start another round of the cycle. (C) Domain composition of proteins involved in RNA interference.

Figure 2. Domains involved in nucleic acid binding in RNAi

(A) PAZ domain of human Argonaute eIF2c1 (PDB: 1SI3). (B) Comparison of nucleic acid binding by PAZ domains from eIF2c1 (green) and T. thermophilus Ago (PDB: 3DLH) (blue). The nucleic acid from each structure is shown in cartoon representation (light green for eIF2c1 structure and purple blue for T. thermophilus Ago structure). The conserved tyrosine that binds the nucleic acid backbone is shown in ball-and-stick representation. (C) dsRBD (ds-RNA-binding domain) from X. leavis RNA-binding protein A bound to a dsRNA duplex (PDB: 1DI2). (D) dsRBD from Rnt1 bound to a tetraloop-stem RNA (PDB: 1T4L). (E) Mid domain from A. fulgidus PIWI protein bound to ssRNA (PDB: 1YTU). The C-terminal carboxylate is shown in red and the metal ion coordinating the 5’-phosphate of the RNA in green. Conserved residues stabilizing the 5’ end of the RNA are shown in balls-and-sticks. (F) Close-up view of the interactions stabilizing the 5’ end of the RNA.

Figure 3. RNases III

(A) A. aeolicus RNase III – intermolecular homodimer. The two endoNDs from two subunits are shown in orange and pink with the central perpendicular helices shown in darker colors. The residues forming the active site are shown in colored balls-and-sticks and those forming the hydrophobic core of the dimer interface in white balls-and-sticks. (B) G. intestinalis Dicer – the intramolecular dimerization of two endoND domains. The linker domain inserted between them is shown in light grey. (C) The structure of A. aeolicus RNase III in complex with dsRNA (PDB: 2NUF). dsRBDs from each dimer subunit are in purple and blue. The RNA is shown in red. (D) The structure of G. intestinalis Dicer (PDB: 2FFL). The PAZ domain is shown in green and the platform domain in light blue. dsRNA modeled to interact with the endoND and PAZ domains is shown in yellow.

Figure 4. Argonautes

(A) The RNase H-like PIWI domain of P. furiosus Argonaute. The central β-sheet is shown in red and the catalytic residues – two aspartates and a histidine as balls-and-sticks. The site of an ~80 residue insertion into the RNase H fold, which is mainly composed of β-strands, is indicated with a gray dashed line. (B) Comparison of apo P. furiosus Ago structure (left, PDB: 1Z25) and T. thermophilus Ago in complex with 21-nt ss DNA (right, PDB: 3DLH). The protein structures were superimposed using the positions of the active site residues in PIWI (RNase H-like) domain. The central β-sheets of nucleic acid binding domains are shown in color (N-ter - blue, PAZ -green, Mid - yellow, and RNase H-like in red) and the differences in their positions between the two structures are indicated with arrows. (C) Structure of T. thermophilus Ago complexed with an RNA/DNA hybrid (PDB: 3F73). The nucleic acid is shown in cartoon representation (RNA in pink and DNA in light blue). The central β-sheets of each domain are colored as in B. The active site carboxylates are shown in ball-and-sticks.