Phillip D. Zamore

RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Small silencing RNAs: an expanding universe.

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.

Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes

In the Drosophila and mammalian RNA interference pathways, siRNAs direct the protein Argonaute2 (Ago2) to cleave corresponding mRNA targets, silencing their expression. Ago2 is the catalytic component of the RNAi enzyme complex, RISC. For each siRNA duplex, only one strand, the guide, is assembled into the active RISC; the other strand, the passenger, is destroyed. An ATP-dependent helicase has been proposed first to separate the two siRNA strands, then the resulting single-stranded guide is thought to bind Ago2. Here, we show that Ago2 instead directly receives the double-stranded siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand, thereby liberating the single-stranded guide. For siRNAs, virtually all RISC is assembled through this cleavage-assisted mechanism. In contrast, passenger-strand cleavage is not important for the incorporation of miRNAs that derive from mismatched duplexes.

SEQUENCE-SPECIFIC INHIBITION OF SMALL RNA FUNCTION

Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2′-O-methyl oligonucleotides can act as irreversible, stoichiometric inhibitors of small RNA function. We show that a 2′-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2′-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2′-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein–RNA complex. Thus, we demonstrate that 2′-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.

microPrimer: the biogenesis and function of microRNA.

Discovered in nematodes in 1993, microRNAs (miRNAs) are non-coding RNAs that are related to small interfering RNAs (siRNAs), the small RNAs that guide RNA interference (RNAi). miRNAs sculpt gene expression profiles during plant and animal development. In fact, miRNAs may regulate as many as one-third of human genes. miRNAs are found only in plants and animals, and in the viruses that infect them. miRNAs function very much like siRNAs, but these two types of small RNAs can be distinguished by their distinct pathways for maturation and by the logic by which they regulate gene expression.

Diversifying microRNA sequence and function

MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are only now beginning to understand how they are generated, assembled into functional complexes and destroyed. Various mechanisms have now been identified that regulate miRNA stability and that diversify miRNA sequences to create distinct isoforms. The production of different isoforms of individual miRNAs in specific cells and tissues may have broader implications for miRNA-mediated gene expression control. Rigorously testing the many discrepant models for how miRNAs function using quantitative biochemical measurements made in vivo and in vitro remains a major challenge for the future.

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5¿ region

MicroRNAs (miRNAs) are ∼22‐nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA‐guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA‐directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3′ end of the miRNA compared to mismatches to the miRNA 5′ region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5′ portion of the small silencing RNA appears crucial regardless of the mode of post‐transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

RNAi: nature abhors a double-strand

In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.

Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein.

microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs f00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.

Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.