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Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis.Significance:Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3′ UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3′ end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Alternative cleavage and polyadenylation (APA) contributes to the diversity of mRNA 3′ ends, affecting post-transcriptional regulation by including or excluding cis-regulatory elements in mRNAs, altering their stability and translational efficiency. While APA analysis has been applied broadly in mixed populations of cells, the heterogeneity of APA among single cells has only recently begun to be explored. We developed an approach we termed scraps (Single Cell RNAPolyA Site Discovery), implemented as a user-friendly, scalable, and reproducible end-to-end workflow, to identify polyadenylation sites at near-nucleotide resolution in single cells using 10X Genomics and other TVN-primed single-cell RNA-seq (scRNA-seq) libraries. Our approach, which performs best with long (>100bp) read 1 sequencing and paired alignment to the genome, is both unbiased relative to existing methods that utilize only read 2 and recovers more sites at higher resolution, despite the reduction in read quality observed on most modern DNA sequencers following homopolymer stretches. For libraries sequenced without long read 1, we implement a fallback approach using read 2-only alignments that performs similarly to our optimal approach, but recovers far fewer polyadenylation sites per experiment. scraps also enables assessment of internal priming capture events, which we demonstrate occur commonly but at higher frequency during apoptotic 3′ RNA decay. We also provide an R package, scrapR, that integrates the results of the scaps pipeline with the popular Seruat single-cell analysis package. Refinement and expanded application of these approaches will further clarify the role of APA in single cells, as well as the effects of internal priming on expression measurements in scRNA-seq libraries.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. Significance: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO‐expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO‐mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO(−/−) mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus‐scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of CEP131, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.

While viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the dLN and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. These findings uncover a previously unrecognized arbovirus scavenging role for LECs and improve our mechanistic understanding of viremia control during arboviral infections.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3′ UTR of an mRNA, thus determining much of its post-transcriptional fate. Here, using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses a AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes that show evidence of alternative polyadenylation. These results suggest that despite pared down cleavage and polyadenylation machinery, 3′ end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Control of centrosome assembly is critical for cell division, intracellular trafficking and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule cytoskeleton, centrosome and centriolar satellites. SON is required for the proper splicing and expression ofCEP131which encodes a major centriolar satellite protein and is required to organize the trafficking and microtubule network around the centrosomes. This study highlights the importance of the distinct microtubule trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development.

Live, attenuated vaccines generate humoral and cellular immune memory, increasing the duration of protective immune memory. We previously found that antigens derived from vaccination or viral infection persist within lymphatic endothelial cells (LECs) beyond the clearance of the infection, a process we termed “antigen archiving”. Technical limitations of fluorescent labeling have precluded a complete picture of antigen archiving across cell types in the lymph node. We developed a “molecular tracking device” to follow the distribution, acquisition, and retention of antigen in the lymph node. We immunized mice with an antigen conjugated to a nuclease-resistant DNA tag and used single-cell mRNA sequencing to quantify its abundance in lymph node hematopoietic and non-hematopoietic cell types. At early and late time points after vaccination we found antigen acquisition by dendritic cell populations (DCs), associated expression of genes involved in DC activation and antigen processing, and antigen acquisition and archiving by LECs as well as unexpected stromal cell types. Variable antigen levels in LECs enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention. Molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigens and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

BackgroundIn single-cell RNA sequencing (scRNA-seq) analysis, assignment of likely cell types remains a time-consuming, error-prone, and biased process. Current packages for identity assignment use limited types of reference data, and often have rigid data structure requirements. As such, a more flexible tool, capable of handling multiple types of reference data and data structures, would be beneficial.FindingsTo address difficulties in cluster identity assignment, we developed the clustifyr R package. The package leverages external datasets, including gene expression profiles from scRNA-seq, bulk RNA-seq, microarray expression data, and/or signature gene lists, to assign likely cell types. We benchmark various parameters of a correlation-based approach, and also implement a variety of gene list enrichment methods. By providing tools for exploratory data analysis, we demonstrate the feasibility of a simple and effective data-driven approach for cell type assignment in scRNA-seq cell clusters.Conclusionsclustifyr is a lightweight and effective cell type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr

Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5′ paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5′ ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from2 kb/min to 1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II (pol II). The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes.

Summary In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3′ flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5′ pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.

The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5'PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes, suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

Multiple lines of evidence indicate that the AP-2 transcription factor family has an important regulatory function in human craniofacial development. Notably, mutations in TFAP2A, the gene encoding AP-2α, have been identified in patients with Branchio-Oculo-Facial Syndrome (BOFS). BOFS is an autosomal-dominant trait that commonly presents with facial clefting, eye defects and branchial skin anomalies. Examination of multiple cases has suggested either simple haploinsufficiency or more complex genetic causes for BOFS, especially as the clinical manifestations are variable, with no clear genotype–phenotype correlation. Mutations occur throughout TFAP2A, but mostly within conserved sequences within the DNA contact domain of AP-2α. However, the consequences of the various mutations for AP-2α protein function have not been evaluated. Therefore, it remains unclear if all BOFS mutations result in similar changes to the AP-2α protein or if they each produce specific alterations that underlie the spectrum of phenotypes. Here, we have investigated the molecular consequences of the mutations that localize to the DNA-binding region. We show that although individual mutations have different effects on DNA binding, they all demonstrate significantly reduced transcriptional activities. Moreover, all mutant derivatives have an altered nuclear:cytoplasmic distribution compared with the predominantly nuclear localization of wild-type AP-2α and several can exert a dominant-negative activity on the wild-type AP-2α protein. Overall, our data suggest that the individual TFAP2A BOFS mutations can generate null, hypomorphic or antimorphic alleles and that these differences in activity, combined with a role for AP-2α in epigenetic events, may influence the resultant pathology and the phenotypic variability.

Summary Anabaena variabilis ATCC 29413 fixes nitrogen in specialized cells called heterocysts using either a Mo-nitrogenase or a V-nitrogenase. V-nitrogenase structural genes, vnfDGK, as well as vnfEN form an operon with ava4025, located upstream of vnfDG that isrepressedbyfixednitrogenandbyMo.Theava4025vnfDGKEN operon is under the control of a Morepressible promoter located nearly 600 bp upstream of ava4025. Levels of vnfDG transcript were about 500-fold higher than ava4025, the first gene of the operon. This may be the result of RNA processing at a site 87 bp upstream of vnfDG that was initially identified as the transcription start site. A strain with a deletion in the coding region of ava4025 grew diazotrophically with Mo or with V. Two similar proteins, VnfR1 and VnfR2, whose genes are located some distance from the ava4025-vnfDGKEN operon, each repressed transcription from the ava4025-vnfDGKEN promoter and a mutant lacking both VnfR1 and VnfR2 made the V-nitrogenase in the presence of Mo. Overexpression of the V-nitrogenase in the double vnfR1 vnfR2 mutant resulted in decreased activity of the Mo-nitrogenase.VnfR1boundspecifically,invitro,toa region upstream of the ava4025 promoter.

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs. DOI: http://dx.doi.org/10.7554/eLife.08146.001, eLife digest Many viruses store their genetic information in the form of strands of ribonucleic acid (RNA), which contain building blocks called nucleotides. Once inside an infected cell, the virus hijacks the cellular structures that build proteins (called ribosomes), which forces the cell to start making viral proteins. Many RNA viruses manipulate the cell’s ribosomes using RNA elements called Internal Ribosome Entry Sites, or IRESs. In a family of viruses called Dicistroviridae, which infect a number of insects, a section of the IRES RNA binds directly to the ribosome. Proteins called elongation factors then trigger a series of events that lead to the cell starting to make the viral proteins. By mutating the RNA of many different Dicistroviridae viruses that infect a variety of invertebrates, Ruehle et al. have now investigated how a particular loop in the structure of the IRES helps to make cells build the viral proteins. This loop is flexible, and interacts with the ribosome to enable the IRES to move through the ribosome. Mutations that shorten the loop or alter the sequence of nucleotides in the loop prevent the occurrence of two of the steps that need to occur for the cell to make viral proteins. Both of these steps depend on elongation factors. Determining how the entire IRES might change shape as it moves through the ribosome is an important next step, since the ribosome is exquisitely sensitive to the shape and motions of its binding partners. DOI: http://dx.doi.org/10.7554/eLife.08146.002

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.