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The article traces significant developments and possibilities in human molecular genetics and genomics. Topics discussed include the transition of emphasis of genomic research from the human genetic code fundamentals to code variation, role of the discovery of genes for mendelian diseases in its drug treatments and gene therapies, and the importance of immediate and free access to data on genomes, transcriptomes and chromatin marks in genomic research.

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection. [ABSTRACT FROM AUTHOR]

The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems now functionalized as biotechnologies. We discuss the rapidly evolving landscape of CRISPR-Cas applications, from gene editing to transcriptional regulation, imaging, and diagnostics. Continuing functional dissection and an expanding landscape of applications position CRISPR-Cas tools at the cutting edge of nucleic acid manipulation that is rewriting biology. [ABSTRACT FROM AUTHOR]

This Genomics and Precision Health article explains the use of CRISPR genome editing in the treatment and prevention of disease. [ABSTRACT FROM AUTHOR]

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine-edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology. [ABSTRACT FROM AUTHOR]

Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90–99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The bacterial Csm complex efficiently knocks down eukaryotic nuclear and cytoplasmic RNAs. [ABSTRACT FROM AUTHOR]

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases. [ABSTRACT FROM AUTHOR]

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins. [ABSTRACT FROM AUTHOR]

DNA encodes the information necessary for life, but sometimes this code also leads to disease. Scientists have long envisioned the ability to change the DNA sequence in cells to correct disease-causing information. A technol- ogy known as CRISPR now enables precise rewriting of DNA sequences, offering unparalleled potential for altering the code of life in human beings as well as other organisms. CRISPR technology holds the promise of curing genetic disease and provides methods to reshape the biosphere for the benefit of human societies and the environment. However, along with these enormous opportunities come safety risks and ethical concerns. This article discusses the uses of CRISPR technology, its potential applications, and the actions we must take to prepare for future developments. [ABSTRACT FROM AUTHOR]

The article discusses the fields of molecular biology, genetics, and genomics are useful to edit specific base pairs or segments of DNA in cells and living organisms. Topics include advent of clustered regularly interspaced short palindromic repeat genome editing, coupled with advances in computing; and diagnose human diseases and predict individual susceptibility based on personal genetics with alter genes responsible for plant traits, transforming agricultural research and plant breeding.

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. [ABSTRACT FROM AUTHOR]

Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control. [ABSTRACT FROM AUTHOR]

Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding. [ABSTRACT FROM AUTHOR]

Many bacteria and archaea possess an adaptive immune system consisting of repetitive genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Similar to RNAi pathways in eukaryotes, CRISPR–Cas systems require small RNAs for sequence-specific detection and degradation of complementary nucleic acids. Cas5 and Cas6 enzymes have evolved to specifically recognize and process CRISPR-derived transcripts into functional small RNAs used as guides by interference complexes. Our detailed understanding of these proteins has led to the development of several useful Cas6-based biotechnological methods. Here, we review the structures, functions, mechanisms, and applications of the enzymes responsible for CRISPR RNA (crRNA) processing, highlighting a fascinating family of endonucleases with exquisite RNA recognition and cleavage activities. [ABSTRACT FROM AUTHOR]

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. [ABSTRACT FROM AUTHOR]

Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral infection. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha, precursor microRNAs (pre-miRNAs) are transported to the cytoplasm, where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins, and of RNA-bound complexes, have offered exciting insights into the mechanisms operating at the heart of RNA-silencing pathways. [ABSTRACT FROM AUTHOR]

Summary: Cell survival in changing environments requires appropriate regulation of gene expression, including posttranscriptional regulatory mechanisms. From reporter gene studies in glucose-starved yeast, it was proposed that translationally silenced eukaryotic mRNAs accumulate in P bodies and can return to active translation. We present evidence contradicting the notion that reversible storage of nontranslating mRNAs is a widespread and general phenomenon. First, genome-wide measurements of mRNA abundance, translation, and ribosome occupancy after glucose withdrawal show that most mRNAs are depleted from the cell coincident with their depletion from polysomes. Second, only a limited subpopulation of translationally repressed transcripts, comprising fewer than 400 genes, can be reactivated for translation upon glucose readdition in the absence of new transcription. This highly selective posttranscriptional regulation could be a mechanism for cells to minimize the energetic costs of reversing gene-regulatory decisions in rapidly changing environments by transiently preserving a pool of transcripts whose translation is rate-limiting for growth. [Copyright &y& Elsevier]

A new series of genetic screens begins to illuminate the interaction between influenza virus and the infected cell. [ABSTRACT FROM AUTHOR]

Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs), and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi). [Copyright &y& Elsevier]

Transmission of influenza viruses into the human population requires surmounting barriers to cross-species infection. Changes in the influenza polymerase overcome one such barrier. Viruses isolated from birds generally contain polymerases with the avian signature glutamic acid at amino acid 627 in the PB2 subunit. These polymerases display restricted activity in human cells. An adaptive change in this residue from glutamic acid to the human-signature lysine confers high levels of polymerase activity in human cells. This mutation permits escape from a species-specific restriction factor that targets polymerases from avian viruses. A 2009 swine-origin H1N1 influenza A virus recently established a pandemic infection in humans, even though the virus encodes a PB2 with the restrictive glutamic acid at amino acid 627. We show here that the 2009 H1N1 virus has acquired second-site suppressor mutations in its PB2 polymerase subunit that convey enhanced polymerase activity in human cells, Introduction of this polymorphism into the PB2 subunit of a primary avian isolate also increased polymerase activity and viral replication in human and porcine cells. An alternate adaptive strategy has also been identified, whereby introduction of a human PA subunit into an avian polymerase overcomes restriction in human cells. These data reveal a strategy used by the 2009 Hi Ni influenza A virus and identify other pathways by which avian and swine-origin viruses may evolve to enhance replication and potentially pathogenesis, in humans. [ABSTRACT FROM AUTHOR]

In plants, SGS3 and RNA-dependent RNA polymerase 6 (RDR6) are required to convert single- to double-stranded RNA (dsRNA) in the innate RNAi-based antiviral response and to produce both exogenous and endogenous short-interfering RNAs. Although a role for RDR6-catalysed RNA-dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA-binding protein with unexpected substrate selectivity favouring 5′-overhang-containing dsRNA. The conserved XS and coiled-coil domains are responsible for RNA-binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi-based host immune response, is a dsRNA-binding protein with similar specificity to SGS3. In competition-binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA-mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs. [ABSTRACT FROM AUTHOR]

In eukaryotes, small non-coding RNAs regulate gene expression, helping to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. These pathways involve two specialized ribonucleases that control the production and function of small regulatory RNAs. The enzyme Dicer cleaves double-stranded RNA precursors, generating short interfering RNAs and microRNAs in the cytoplasm. These small RNAs are transferred to Argonaute proteins, which guide the sequence-specific silencing of messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation. The molecular structures of Dicer and the Argonaute proteins, free and bound to small RNAs, have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways. [ABSTRACT FROM AUTHOR]

In this issue, Ahl et al. (2015) and Doucet et al. (2015) illuminate structural and functional features of substrates that promote integration of RNA molecules into the human genome by LINE retrotransposons, contributing to the ∼50% of the human genome that has been colonized by mobile genetic elements. [ABSTRACT FROM AUTHOR]

The crystal structure of the enzyme Dicer from Giardia intestinalis was solved to 3.3 Å resolution by MAD using crystals belonging to space group P21212 [Macrae et al. (2006), Science, 311, 195–198]. These crystals were derived from crystals that diffracted X-rays to 3.0 Å resolution but were refractory to structure determination because they were twinned. It is shown here that the original Dicer crystals represent an unusual case of perfect pseudo-merohedral twinning of orthorhombic crystals. Before the twinning problem was overcome, it was possible to calculate a low-resolution electron-density map in space group P41 that was used to build a partial molecular model. Experimental phases were sufficient to identify heavy-atom sites that indicated space-group inconsistency, leading to identification of the true space group. This information guided the search for different crystallization conditions that yielded untwinned crystals and ultimately a fully interpretable electron-density map. [ABSTRACT FROM AUTHOR]

Ribonuclease III (RNase III) enzymes occur ubiquitously in biology and are responsible for processing RNA precursors into functional RNAs that participate in protein synthesis, RNA interference and a range of other cellular activities. Members of the RNase III enzyme family, including Escherichia coli RNase III, Rnt1, Dicer and Drosha, share the ability to recognize and cleave double-stranded RNA (dsRNA), typically at specific positions or sequences. Recent biochemical and structural data have shed new light on how RNase III enzymes catalyze dsRNA hydrolysis and how substrate specificity is achieved. A major theme emerging from these studies is that accessory domains present in different RNase III enzymes are the key determinants of substrate selectivity, which in turn dictates the specialized biological function of each type of RNase III protein. [Copyright &y& Elsevier]

Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis. [ABSTRACT FROM AUTHOR]

The article informs that CRISPR-Cas9 gene-edited T cells show safety and long-term engraftment in humans. It mentions that production of engineered cell therapies involves transduction of isolated patient T cells with a disarmed virus to express a receptor recognizing an antigen present on the outside of cancer cells.

Ribosome biogenesis in eukaryotic organisms involves the coordinated assembly of 78 ribosomal proteins onto the four ribosomal RNAs, mediated by a host of trans-acting factors whose specific functions remain largely unknown. The essential GTPase Bms1, the putative endonuclease Rcl1 and the essential U3 small nucleolar RNA form a stable subcomplex thought to control an early step in the assembly of the 40S ribosomal subunit. Here, we provide a complete thermodynamic analysis of GTP-dependent subcomplex formation, revealing strong thermodynamic coupling of Rcl1, U3 small nucleolar RNA and GTP binding to Bms1 that is eliminated in the presence of GDP. The results suggest that Rcl1 activates Bms1 by promoting GDP/GTP exchange, analogous to ribosome-promoted nucleotide exchange within translation elongation factor EF-G. These and other data unveil thermodynamic similarities between Bms1 and the subgroup of GTPases involved in translation, providing evidence that parts of the ribosome assembly machinery may have evolved from the translation apparatus. This quantitative description of an early and essential step in pre-ribosome assembly provides a framework for elucidating the network of interactions between the Bms1 subcomplex and additional factors involved in ribosome biogenesis. [Copyright &y& Elsevier]

The article focuses on the chemical insight into the function of small molecules, the building blocks, effectors and probes of macromelecular structure and function. They offer a common connection between the fields of chemical, structure and cell biology. The composition and role of huge RNA molecules are also discussed.

Evolution has resoundingly favored protein enzymes over RNA-based catalysts, yet ribozymes occupy important niches in modern cell biology that include the starring role in catalysis of protein synthesis on the ribosome. Recent results from structural and biochemical studies show that natural ribozymes use an impressive range of catalytic mechanisms, beyond metalloenzyme chemistry and analogous to more chemically diverse protein enzymes. These findings make it increasingly possible to compare details of RNA- and protein-based catalysis. [ABSTRACT FROM AUTHOR]

RNA plays a direct role in a variety of cellular activities, and in many cases its biological function is conferred by the RNA three-dimensional structure. X-ray crystallography is the method of choice for determining high resolution structures of large RNA molecules, and can also be used to compare related RNAs and identify conformational changes that may accompany biochemical activity. However, crystallization remains the rate-limiting step in RNA structure determination due to the difficulty in obtaining well-ordered crystals for X-ray diffraction analysis. Several approaches to sample preparation, crystallization, and crystal handling are presented that have been used successfully in the structure determination of RNA and RNA–protein complexes in our laboratory, and should be generally applicable to RNAs in other systems. [Copyright &y& Elsevier]

The signal recognition particle (SRP) directs integral membrane and secretory proteins to the cellular protein translocation machinery during translation. The SRP is an evolutionarily conserved RNA-protein complex whose activities are regulated by GTP hydrolysis. Recent structural investigations of SRP functional domains and interactions provide new insights into the mechanisms of SRP activity in all cells, leading toward a comprehensive understanding of protein trafficking by this elegant pathway. [ABSTRACT FROM AUTHOR]

Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex. The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA. The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism. To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs. Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV. However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case. Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA. Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron. These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP. [ABSTRACT FROM AUTHOR]

Examines the structural and energetic analysis of metal ions essential to signal recognition particle domain assembly. Performance of structural analysis of RNA and protein components; Use of binding affinity assays and x-ray crystallography; Analysis of the specificity and energetic contributions of mono and divalent metal ions.

Functional RNAs often form compact structures characterized by closely packed helices. Crystallographic analysis of several large RNAs revealed a prevalent interaction in which unpaired adenosine residues dock into the minor groove of a receptor helix. This A-minor motif, potentially the most important element responsible for global RNA architecture, has also been suggested to contribute to the fidelity of protein synthesis by discriminating against nearcognate tRNAs on the ribosome. The specificity of A-minor interactions is fundamental to RNA tertiary structure formation, as well as to their proposed role in translational accuracy. To investigate A-minor motif specificity, we analyzed mutations in an A-minor interaction within the Tetrahymena group I self-splicing intron. Thermodynamic and x-ray crystallographic results show that the A-minor interaction strongly prefers canonical base pairs over base mismatches in the receptor helix, enabling RNA interhelical packing through specific recognition of Watson-Crick minor groove geometry. [ABSTRACT FROM AUTHOR]

Explores the role of the RNA molecules, ribozymes in how life began on Earth. Catalysis based on RNA; Information on self-splicing introns; Hepatitis delta virus and hairpin ribozymes.

Examines the structure and function of the eukaryotic ribosome. Emphasis of the catalysts of peptide bond formation; Combination of genetic, biochemical and structural approaches; Aspects of ribosome biogenesis.

Group II self-splicing introns catalyze autoexcision from precursor RNA transcripts by a mechanism strikingly similar to that of the spliceosome, an RNA-protein assembly responsible for splicing together the protein-coding parts of most eukaryotic pre-mRNAs. Splicing in both cases initiates via nucleophilic attack at the 5′ splice site by the 2′ OH of a conserved intron adenosine residue, creating a branched (lariat) intermediate. Here, we describe the crystal structure at 3.0 Å resolution of a 70-nucleotide RNA containing the catalytically essential domains 5 and 6 of the yeast ai5γ/ group II self-splicing intron, revealing an unexpected two-nucleotide bulged structure around the branchpoint adenosine in domain 6. [ABSTRACT FROM AUTHOR]

The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron. [ABSTRACT FROM AUTHOR]

Compares the structures and possible catalytic mechanisms of small self-cleaving RNAs. Roles of divalent metal ions in catalysis; Biological function of hepatitis delta virus ribozyme; Comparative organization of catalytic RNAs.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. [ABSTRACT FROM AUTHOR]

An RNA fold is the result of packing together two or more coaxial helical stacks. To date, four RNA folds have been determined at near-atomic resolution by X-ray crystallography: transfer RNA, the hammerhead ribozyme, the P4–P6 domain of the Tetrahymena group I intron, and the hepatitis delta virus ribozyme. All four folds result in RNAs that are considerably more compact than isolated A-form duplexes. These structures illustrate, to varying degrees, three modes of fold stabilization: association of complementary molecular surfaces, stabilization of close RNA packing by binding of cations, and stabilization through pseudoknotting. [ABSTRACT FROM AUTHOR]

Examines how the P4-P6 domain directs higher order folding of the Tetrahymena ribozyme core. Interface of proposed structural domain; Solvent accessibility of separate fragments; Analysis of structural features; Tertiary interaction of the region.

Reviews the molecular basis of RNA-duplex recognition with a globular ribonucleic acid (RNA) structure. Secondary structure of RNA; Chemical group that distinguishes RNA from deoxyribonucleic acid (DNA); Demonstration of the importance of the noncanonical base pairs in RNA-duplex recognition; Binding of the GAAA loop into the tetraloop receptor; Recognition of the P4 helix by the A-rich bulge; Other potential motifs for packing RNA helices.