A retrovirus packages nascent host noncoding RNAs from a novel surveillance pathway.

Although all retroviruses recruit host cell RNAs into virions, both the spectrum of RNAs encapsidated and the mechanisms by which they are recruited remain largely unknown. Here, we used high-throughput sequencing to obtain a comprehensive description of the RNAs packaged by a model retrovirus, murine leukemia virus. The major encapsidated host RNAs are noncoding RNAs (ncRNAs) and members of the VL30 class of endogenous retroviruses. Remarkably, although Moloney leukemia virus (MLV) assembles in the cytoplasm, precursors to specific tRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) are all enriched in virions. Consistent with their cytoplasmic recruitment, packaging of both pre-tRNAs and U6 snRNA requires the nuclear export receptor Exportin-5. Adenylated and uridylated forms of these RNAs accumulate in cells and virions when the cytoplasmic exoribonuclease DIS3L2 and subunits of the RNA exosome are depleted. Together, our data reveal that MLV recruits RNAs from a novel host cell surveillance pathway in which unprocessed and unneeded nuclear ncRNAs are exported to the cytoplasm for degradation.

HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle.

N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.

Sensitive and quantitative probing of pseudouridine modification in mRNA and long noncoding RNA.

Pseudouridine (Ψ) is the most abundant RNA modification in cellular RNA present in tRNA/rRNA/snRNA and also in mRNA and long noncoding RNA (lncRNA). Elucidation of Ψ function in mRNA/lncRNA requires mapping and quantitative assessment of its modification fraction at single-base resolution. The most widely used Ψ mapping method for mRNA/lncRNA relies on its reaction with N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC), forming an adduct with the Ψ base in RNA that is detectable by reverse transcription (RT) stops. However, this method has not produced consistent Ψ maps in mRNAs; furthermore, available protocols do not lend confidence to the estimation of Ψ fraction at specific sites, which is a crucial parameter for investigating the biological relevance of mRNA modifications. Here we develop a quantitative RT-PCR based method that can detect and quantify the modification fraction of target Ψ sites in mRNA/lncRNA, termed CMC-RT and ligation assisted PCR analysis of Ψ modification (CLAP). The method still relies on RT stop at a CMC-Ψ site, but uses site-specific ligation and PCR to generate two distinct PCR products in the same sample, corresponding to the modified and unmodified site, that are visualized by gel electrophoresis. CLAP not only requires a small amount of cellular RNA to validate Ψ sites but also determines the Ψ fraction semiquantitatively at target sites in mRNA/lncRNA. We determined the Ψ status of four mRNA sites and one lncRNA site whose modification fractions range from 30% to 84% in three human cell lines. Our method enables precise mapping and assessment of Ψ modification levels in low abundance cellular RNAs.

Analysis of the human immunodeficiency virus-1 RNA packageome.

All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response.

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.

A large-scale complex haploinsufficiency-based genetic interaction screen in Candida albicans: analysis of the RAM network during morphogenesis.

The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans.Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1(cbk1Δ/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on SpiderMedium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced,yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyperactivation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKA­regulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM­pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI­based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation.

A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth.

Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1Delta mutants are noninvasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1Delta mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.

Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

Host RNA Packaging by Retroviruses: A Newly Synthesized Story.

A fascinating aspect of retroviruses is their tendency to nonrandomly incorporate host cell RNAs into virions. In addition to the specific tRNAs that prime reverse transcription, all examined retroviruses selectively package multiple host cell noncoding RNAs (ncRNAs). Many of these ncRNAs appear to be encapsidated shortly after synthesis, before assembling with their normal protein partners. Remarkably, although some packaged ncRNAs, such as pre-tRNAs and the spliceosomal U6 small nuclear RNA (snRNA), were believed to reside exclusively within mammalian nuclei, it was demonstrated recently that the model retrovirus murine leukemia virus (MLV) packages these ncRNAs from a novel pathway in which unneeded nascent ncRNAs are exported to the cytoplasm for degradation. The finding that retroviruses package forms of ncRNAs that are rare in cells suggests several hypotheses for how these RNAs could assist retrovirus assembly and infectivity. Moreover, recent experiments in several laboratories have identified additional ways in which cellular ncRNAs may contribute to the retrovirus life cycle. This review focuses on the ncRNAs that are packaged by retroviruses and the ways in which both encapsidated ncRNAs and other cellular ncRNAs may contribute to retrovirus replication.

Genetic networks inducing invasive growth in Saccharomyces cerevisiae identified through systematic genome-wide overexpression.

The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This growth transition has been studied extensively as a model signaling system with similarity to processes of hyphal development that are linked with virulence in related fungal pathogens. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. Here, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. This cohort includes 79 genes lacking previous phenotypic characterization. Pathway enrichment analysis of the gene set identifies networks mediating mitogen-activated protein kinase (MAPK) signaling and cell cycle progression. In particular, overexpression screening suggests that nuclear export of the osmoresponsive MAPK Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. Through epistasis and deletion studies, we also identified genetic relationships with the G2 cyclin Clb2p and phenotypes in filamentation induced by S-phase arrest. In sum, this work presents a unique and informative resource toward understanding the breadth of genes and pathways that collectively constitute the molecular basis of filamentation.