David W. Taylor

A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity

No Dicer for Me MicroRNAs (miRNAs) are small noncoding RNAs found in most eukaryotes. Most are processed from primary transcripts in the nucleus by the microprocessor enzyme complex, which includes the nuclease Drosha, with a small number being generated by the messenger RNA splicing machinery. All pre-miRNAs are then exported into the cytoplasm where they are cleaved further by a second nuclease, Dicer, into the mature, functional miRNA. Cifuentes et al. (p. 1694, published online 6 May), now show that in a Dicer mutant fish at least one miRNA, miR-451, is still formed from pre-miR-451. The processing of pre-miR-451 requires the slicing activity of another protein in the miRNA pathway, Argonaute2. The unusual secondary structure of the pre-miR-451 determines its noncanonical processing pathway, which suggests that other miRNAs might also be processed in this way. The unusual secondary structure of a precursor microRNA determines its noncanonical processing. Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation.

Introduction Bacteria and archaea defend themselves against invasive DNA using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) loci and CRISPR-associated (Cas) genes. In association with Cas proteins, small CRISPR RNAs (crRNAs) guide the detection and cleavage of complementary DNA sequences. Type II CRISPR systems employ the RNA-guided endonuclease Cas9 to recognize and cleave double-stranded DNA (dsDNA) targets using conserved RuvC and HNH nuclease domains. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. Recently, the biochemical properties of Cas9–guide RNA complexes have been harnessed for various genetic engineering applications and RNA-guided transcriptional control. Despite these ongoing successes, the structural basis for guide RNA recognition and DNA targeting by Cas9 is still unknown. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. (A) Crystal structures of S. pyogenes (SpyCas9) and A. naeslundii (AnaCas9) Cas9 proteins. (B) Left: Negative-stain EM reconstructions of apo-SpyCas9 (top) and SpyCas9-RNA-target DNA complex (bottom) show that nucleic acid binding causes a reorientation of the nuclease (blue) and α-helical (gray) lobes in SpyCas9. Right: Cartoon representations of the structures. tracrRNA, trans-activating crRNA. Rationale To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) were determined by x-ray crystallography. Crosslinking of target DNA containing 5-bromodeoxyuridines was conducted to identify PAM-interacting regions in SpyCas9. To test functional interactions with nucleic acid ligands, structure-based mutant SpyCas9 proteins were assayed for endonuclease activity with radiolabeled oligonucleotide dsDNA targets, and target DNA binding was monitored by electrophoretic mobility shift assays. To compare conformations of Cas9 in different states of nucleic acid binding, three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas9:RNA:DNA were obtained by negative-stain single-particle electron microscopy. Guide RNA and target DNA positions were determined with streptavidin labeling. Exonuclease protection assays were carried out to determine the extent of Cas9–target DNA interactions. Results The 2.6 Å–resolution structure of apo-SpyCas9 reveals a bilobed architecture comprising a nuclease domain lobe and an α-helical lobe. Both lobes contain conserved clefts that may function in nucleic acid binding. Photocrosslinking experiments show that the PAM in target DNA is engaged by two tryptophan-containing flexible loops, and mutations of both loops impair target DNA binding and cleavage. The 2.2 Å–resolution crystal structure of AnaCas9 reveals the conserved structural core shared by all Cas9 enzyme subtypes, and both SpyCas9 and AnaCas9 adopt autoinhibited conformations in their apo forms. The electron microscopic (EM) reconstructions of SpyCas9:RNA and SpyCas9:RNA:DNA complexes reveal that guide RNA binding results in a conformational rearrangement and formation of a central channel for target DNA binding. Site-specific labeling of guide RNA and target DNA define the orientations of nucleic acids in the target-bound complex. Conclusion The SpyCas9 and AnaCas9 structures define the molecular architecture of the Cas9 enzyme family in which a conserved structural core encompasses the two nuclease domains responsible for DNA cleavage, while structurally divergent regions, including the PAM recognition loops, are likely responsible for distinct guide RNA and PAM specificities. Cas9 enzymes adopt a catalytically inactive conformation in the apo state, necessitating structural activation for DNA recognition and cleavage. Our EM analysis shows that by triggering a conformational rearrangement in Cas9, the guide RNA acts as a critical determinant of target DNA binding. Cas9 Solved Clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) loci allow prokaryotes to identify and destroy invading DNA. Not only important to bacteria, the universal value of Cas endonuclease specificity has also resulted in Cas9 being exploited as a tool for genome editing. Jinek et al. (10.1126/science.1247997, published online 6 February) determined the 2.6 and 2.2 angstrom resolution crystal structures of two Cas9 enzymes to reveal a common structural core with distinct peripheral elaborations. The enzymes are autoinhibited, undergo large conformational changes on binding RNA, and have channels lined with basic residues that are candidates for an RNA-DNA binding groove. Based on these and other insights from the structures, this work provides important revelations both for the CRISPR mechanism and for genome editing. Binding of a guide RNA triggers structural changes in a set of DNA-cleaving enzymes. Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

CRISPR Cas9 molecular scissors The CRISPR-associated (Cas) protein Cas9 is a molecular scissor for cutting DNA. The first step in the cutting reaction is the RNA-guided unwinding of the DNA double helix. Jiang et al. determined the structures of Cas9 bound to DNA unwound by the targeting RNA (see the Perspective by Chen and Bailey). Cas9 bends the DNA to allow guide RNA infiltration into the double helix. The two separated DNA strands, one bound to RNA, are subsequently positioned in the dual active sites of the protein for cutting. Science, this issue p. 867; see also p. 811 The CRISPR-Cas9 nuclease helps guide RNA to open the target DNA strands and position them for cutting. [Also see Perspective by Chen and Bailey] Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

Rational design of a split-Cas9 enzyme complex

Significance Bacteria have evolved clustered regularly interspaced short palindromic repeats (CRISPRs) together with CRISPR-associated (Cas) proteins to defend themselves against viral infection. RNAs derived from the CRISPR locus assemble with Cas proteins into programmable DNA-targeting complexes that destroy DNA molecules complementary to the guide RNA. In type II CRISPR-Cas systems, the Cas9 protein binds and cleaves target DNA sequences at sites complementary to a 20-nt guide RNA sequence. This activity has been harnessed for a wide range of genome-engineering applications. This study explores the structural features that enable Cas9 to bind and cleave target DNAs, and the results suggest a way of regulating Cas9 by physical separation of the catalytic domains from the rest of the protein scaffold. Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

Phylogenetic evidence for the herbaceous origin of angiosperms

The ancestral angiosperm is commonly interpreted as an arborescent to shrubby magnolialean with large, multiparted, complex flowers. We examined this hypothesis using a phylogenetic analysis of new and reevaluated characters polarizabled with outgroup comparison. Our cladistic analysis of basal angiosperms placed the nonmagnolialeanChloranthaceae andPiperaceae at the bottom of the tree. We further inferred the probable ancestral states of characters not polarizable with outgroup comparison by examining their distribution among taxa at the base of our cladogram. The sum of ancestral character states suggests that the protoangiosperm was a diminutive, rhizomatous to scrambling perennial herb, with small, simple flowers.

An Aptian Plant with Attached Leaves and Flowers: Implications for Angiosperm Origin

Recent phylogenetic studies and fossil finds support a new view of the ancestral angiosperm. A diminutive fossil angiosperm from the Aptian of Australia has attached leaves, with intermediate pinnate-palmate, low-rank venation, and lateral axes bearing pistillate organs subtended by bracts and bracteoles that are the oldest direct evidence of flowers. A variety of data suggests a similar morphology for the ancestral angiosperm. This hypothesis explains similarities between rhizomatous to herbaceous Magnoliidae and basal monocots, scarcity of early agniosperm wood, and lack of recognition of earlier remains.

RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Early steps of angiosperm–pollinator coevolution

The hypothesis that early flowering plants were insect-pollinated could be tested by an examination of the pollination biology of basal angiosperms and the pollination modes of fossil angiosperms. We provide data to show that early fossil angiosperms were insect-pollinated. Eighty-six percent of 29 extant basal angiosperm families have species that are zoophilous (of which 34% are specialized) and 17% of the families have species that are wind-pollinated, whereas basal eudicot families and basal monocot families more commonly have wind and specialized pollination modes (up to 78%). Character reconstruction based on recent molecular trees of angiosperms suggests that the most parsimonious result is that zoophily is the ancestral state. Combining pollen ornamentation, size, and aperture characteristics and the abundance of single-species pollen clumps of Cenomanian angiosperm-dispersed pollen species from the Dakota Formation demonstrates a dominance of zoophilous pollination (76% versus 24% wind pollination). The zoophilous pollen species have adaptations for pollination by generalist insects (39%), specialized pollen-collecting insects (27%), and other specialized pollinators (10%). These data quantify the presences of more specialized pollination modes during the mid-Cretaceous angiosperm diversification.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference

Significance Bacteria use clustered regularly interspaced short palindromic repeats (CRISPRs) together with CRISPR-associated (Cas) proteins to defend themselves against viral infection. The CRISPR locus contains short segments acquired from viral genomes, and RNAs derived from these segments assemble with Cas proteins into programmable DNA-binding complexes that target DNA molecules complementary to the guide RNA for cleavage. In type I CRISPR-Cas systems, the CRISPR-associated complex for antiviral defense (Cascade) binds to target DNA sequences and then recruits the Cas3 enzyme to repeatedly cleave the bound DNA. In this study, we show how Cascade positions both the DNA and Cas3 to ensure DNA cleavage. In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA–E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Substrate-specific structural rearrangements of human Dicer

Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer–RNA complexes to gain insight into the structural basis for human Dicers substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.