Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is ...required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11, the latter protein binds to RNA and the RNA polymerase II carboxy terminal domain. Here, we used siRNA mediated knock-down and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' un-translated regions (3' UTRs) we observed shifts from promoter proximal regions to the 3' UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells co-sedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.
Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same ...family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) β stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with
Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation.
Understanding multicellular fungal structures is important for designing better strategies against human fungal pathogens. For example, the ability to form multicellular biofilms is a key virulence ...property of the yeast Candida albicans. C. albicans biofilms form on indwelling medical devices and are drug resistant, causing serious infections in hospital settings. Multicellular fungal communities are heterogeneous, consisting of cells experiencing different environments. Heterogeneity is likely important for the phenotypic characteristics of communities, yet it is poorly understood. Here we used colonies of the yeast Saccharomyces cerevisiae as a model fungal multicellular structure. We fractionated the outside colony layers from the cells in the center by FACS, using a Cit1-GFP marker expressed exclusively on the outside. Transcriptomics analysis of the two subpopulations revealed that the outside colony layers are actively growing by fermentative metabolism, while the cells residing on the inside are in a resting state and experience changes to mitochondrial activity. Our data shows several parallels with C. albicans biofilms providing insight into the contributions of heterogeneity to biofilm phenotypes. Hallmarks of C. albicans biofilms - the expression of ribosome and translation functions and activation of glycolysis and ergosterol biosynthesis occur on the outside of colonies, while expression of genes associates with sulfur assimilation is observed in the colony center. Cell wall restructuring occurs in biofilms, and cell wall functions are enriched in both fractions: the outside cells display enrichment of cell wall biosynthesis enzymes and cell wall proteins, while the inside cells express cell wall degrading enzymes. Our study also suggests that noncoding transcription and posttranscriptional mRNA regulation play important roles during growth of yeast in colonies, setting the scene for investigating these pathways in the development of multicellular fungal communities.
Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically ...analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.
Next-generation sequencing (NGS) and its application to RNA (RNA-seq) has opened up multiple aspects of RNA processing to deep transcriptome-wide analysis at nucleotide resolution. This has been ...useful in delineating the transcribed areas of the genome, and in quantitation of RNA isoforms. Such isoforms can diversify the regulatory repertoire of mRNAs. For example, the 3'-end of mRNA can vary in two important ways, in the position chosen for cleavage and polyadenylation, and in the length of the poly(A)-tail. Accordingly, the step-up in resolution made possible by NGS has revealed an unexpectedly high level of alternative polyadenylation (APA). Moreover, it has massively simplified the transcriptome-wide detection of poly(A)-tail length changes. Here we present our approach to the study of 3'-end dynamics using a 3'-focused RNA-seq method called PAT-seq (for poly(A)-test sequencing). The approach records gene expression, APA, and poly(A)-tail changes between transcriptomes to reveal complex interplay between transcriptional and posttranscriptional control mechanisms.
Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches ...to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and unexpected relationships between distinct aspects of gene expression. Translation and polyadenylation are aligned on a global scale with both the lengths and levels of mRNAs: efficiently translated mRNAs have longer poly(A) tails and are shorter, more stable, and more efficiently transcribed on average. Transcription and translation may be independently but congruently optimized to streamline protein production. These rich data sets, all acquired under a standardized condition, reveal a substantial coordination between regulatory layers and provide a basis for a systems-level understanding of multilayered gene-expression programs.
The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct ...support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.
A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and ...adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide.
The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall ...integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.
Identification of multiple histone acylations diversifies transcriptional control by metabolism, but their functions are incompletely defined. Here we report evidence of histone crotonylation in the ...human fungal pathogen Candida albicans. We define the enzymes that regulate crotonylation and show its dynamic control by environmental signals: carbon sources, the short-chain fatty acids butyrate and crotonate, and cell wall stress. Crotonate regulates stress-responsive transcription and rescues C. albicans from cell wall stress, indicating broad impact on cell biology. The YEATS domain crotonylation readers Taf14 and Yaf9 are required for C. albicans virulence, and Taf14 controls gene expression, stress resistance, and invasive growth via its chromatin reader function. Blocking the Taf14 C terminus with a tag reduced virulence, suggesting that inhibiting Taf14 interactions with chromatin regulators impairs function. Our findings shed light on the regulation of histone crotonylation and the functions of the YEATS proteins in eukaryotic pathogen biology and fungal infections.
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•Metabolism and stress responses control histone crotonylation in Candida albicans•The YEATS crotonylation readers Taf14 and Yaf9 are important for Candida virulence•Taf14 participates in stress responses, cell wall, and filamentation pathways•The SCFA crotonate affects transcription and stress susceptibility in Candida
Diverse histone acylations expand functional linkages among metabolism, gene regulation, and cellular responses. Wang et al. report that metabolic and stress cues dynamically control histone crotonylation in the human commensal and pathogen Candida albicans and show the requirement for crotonylation readers in pathogenesis-related pathways and mammalian virulence.
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