Brenton R. Graveley

CRISPR–Cas immunity depends on acquiring spacers that generate functional CRISPR RNA (crRNA) guides. For most RNA-targeting type III systems, this poses a paradox: spacers are captured from DNA but defense requires RNA interaction, thus the system must infer transcriptional relevance of DNA fragments. We compared spacer acquisition profiles for co-occurring type III (RNA-recognizing) and type II (DNA-recognizing) systems in Streptococcus thermophilus during lytic phage infection. Type III spacer acquisition was highly enriched for early phage genes whereas type II spacers were distributed genome wide. High-throughput spacer sequencing showed that the type III early gene bias arose during adaptation, was independent of interference, and peaked immediately downstream of a conserved early phage promoter. This bias for early phage promoters was also observed in natural type III spacers identified in publicly available S. thermophilus genomes. Cloning this early phage promoter and downstream gene into a plasmid recreated the type III-acquisition hotspot. Mutation to disrupt the promoter eliminated the spacer hotspot, while mutation to disrupt translation enhanced it. Thus, type III-A adaptation preferentially samples DNA associated with the earliest phage transcriptional program, enriching for spacers that generate crRNAs targeting early phage RNA.

Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs that play critical roles in guiding 2’-O-methylation (Nm) and pseudouridylation modifications of RNAs. In Drosophila melanogaster , snoRNAs undergo dynamic changes in expression during development. In this study, we identified 239 snoRNAs that are robustly expressed in Drosophila S2 cells, representing 87% of all annotated Drosophila snoRNAs. Given that box C/D snoRNAs guide site-specific 2’-O-methylation (Nm) of RNA, we next characterized the Nm landscape of S2 cells using RibOxi-seq2, a high-throughput approach capable of detecting Nm modifications with single-nucleotide resolution. RibOxi-seq2 revealed 17 Nm sites in 18S rRNA with a 94% concordance to previously reported RiboMeth-Seq data. In 28S rRNA, 30 Nm sites were identified, corresponding to an 71.4% overlap with established references. Additionally, we detected both a known Nm site (Gm74) and a novel site (Um66) in 5.8S rRNA, further validating the sensitivity and specificity of the approach. RibOxi-seq2 further identified Nm sites in small nuclear RNAs (snRNAs), expanding the annotation of modified non-coding RNAs. Additionally, the method revealed Nm modifications within internal regions of mRNAs. In total, we detected Nm modifications in 2,057 unique mRNAs, underscoring the widespread presence of this epitranscriptomic modification in coding transcripts. Strikingly, although we could not identify any snoRNAs predicted to guide the mRNA 2’-O-methylation modifications by canonical mechanisms, we identified strong consensus sequences surrounding many of these mRNA sites. Together, our findings not only expand the known landscape of Nm-modified RNAs but also highlight the robustness of RibOxi-seq2 for transcriptome-wide RNA modification profiling. Collectively, this study presents a comprehensive atlas of snoRNA expression and 2’-O-methylation sites in Drosophila S2 cells, offering valuable insights into the epitranscriptomic landscape orchestrated by snoRNAs.

Reduction in the Indy (I’m not dead yet) gene, a plasma membrane citrate transporter, in Drosophila and its homolog in worms extends lifespan by promoting metabolic homeostasis. Indy reduction delays the onset of aging-associated pathology in the fly midgut, including preservation of intestinal barrier integrity and intestinal stem cell homeostasis. Gut microbiota has broad impacts on host metabolism, health, and aging. Age-related dysbiosis impairs intestinal barrier function and drives mortality. However, the underlying mechanisms that link increased microbial load to frailty and negative effects on health remain mostly unclear. Here we show that Indy heterozygote flies have significantly lower bacterial load and increased diversity during aging compared to controls. However, the presence of the microbiome was not required for Indy lifespan extension, though removal of microbes did enhance the effects of Indy reduction on longevity, suggesting potential interactions between the microbiome and Indy . Indy down-regulation was linked to reduced expression of the JAK/STAT signaling ligands Upd3 and Upd2 in the midgut of young flies, which likely contributes to preserved intestinal stem cell homeostasis. Altogether, our results suggest that Indy reduction impacts microbiome load and composition, which preserves gut homeostasis and extends lifespan through impacts on JAK/STAT signaling pathway. Significance Statement: Indy is a fly homologue of mammalian SLC13A5 (mSLC13A5) plasma membrane citrate transporter, a central metabolic regulator involved in health, longevity, and disease. Reduction of fly Indy gene activity preserves metabolic and intestinal stem cell homeostasis and extends longevity. Gut microbiota impacts host metabolism, health, and aging. Here we show that Indy reduction prevents age-associated increases in bacterial load and expression of the JAK/STAT signaling ligands Upd3, and Upd2, while maintaining microbiome diversity. These changes likely slow activation of epithelial cell turnover in the gut and contribute to downstream lifespan effects. As the role of INDY and microbiome are conserved across organisms, our study provides a framework to study underlying mechanisms of the effects of reduced Indy and the microbiome on health and longevity.

We have compared genome-wide patterns of RNA 2’- O -methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2’- O -methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.

SUMMARY: We have compared genome-wide patterns of RNA 2’- O -methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2’- O -methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.

Type III CRISPR immune systems bind viral or plasmid RNA transcripts and activate Csm3/Cmr4 and Cas10 nucleases to uniquely cleave both invader RNA and DNA, respectively. Additionally, type III effector complexes generate cyclic oligoadenylate (cOA) signaling molecules to activate trans-acting, auxiliary Csm6/Csx1 ribonucleases, previously proposed to be non-specific in their in vivo RNA cleavage preference. Despite extensive in vitro studies, the nuclease requirements of type III systems in their native contexts remain poorly understood. Here we systematically investigated the in vivo roles for immunity of each of the three Streptococcus thermophilus (Sth) type III-A Cas nucleases and cOA signaling by challenging nuclease defective mutant strains with plasmid and phage infections. Our results reveal that RNA cleavage by Csm6 is both sufficient and essential for maintaining wild-type levels of immunity. Importantly, Csm6 RNase activity leads to immunity against even high levels of phage challenge without causing host cell dormancy or death. Transcriptomic analyses during phage infection indicated Csm6-mediated and crRNA-directed preferential cleavage of phage transcripts. Our findings highlight the critical role of Csm6 RNase activity in type III immunity and demonstrate specificity for invader RNA transcripts by Csm6 to ensure host cell survival upon phage infection.

The prevalence of obesity is increasing in older adults and contributes to age-related decline. Caloric restriction (CR) alleviates obesity phenotypes and delays the onset of age-related changes. However, how late in life organisms benefit from switching from a high-(H) to a low-calorie (L) diet is unclear. We transferred male flies from a H to a L (HL) diet or vice versa (LH) at different times during life. Both shifts immediately change fly rate of aging even when applied late in life. HL shift rapidly reduces fly mortality rate to briefly lower rate than in flies on a constant L diet, and extends lifespan. Transcriptomic analysis uncovers that flies aged on H diet have acquired increased stress response, which may have temporal advantage over flies aged on L diet and leads to rapid decrease in mortality rate after HL switch. Conversely, a LH shift increases mortality rate, which is temporarily higher than in flies aged on a H diet, and shortens lifespan. Unexpectedly, more abundant transcriptomic changes accompanied LH shift, including increase in ribosome biogenesis, stress response and growth. These changes reflect protection from sudden release of ROS, energy storage, and use of energy to growth, which all likely contribute to higher mortality rate. As the beneficial effects of CR on physiology and lifespan are conserved across many organisms, our study provides framework to study underlying mechanisms of CR interventions that counteract the detrimental effects of H diets and reduce rate of aging even when initiated later in life.

CRISPR–Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5′ end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5′ end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5′ end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.

Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.

CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I–VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.