The Cross-Regulation Between Set1, Clr4, and Lsd1/2 in Schizosaccharomyces pombe.

Eukaryotic chromatin is organized into either silenced heterochromatin or relaxed euchromatin regions, which controls the accessibility of transcriptional machinery and thus regulates gene expression. In fission yeast, Schizosaccharomyces pombe, Set1 is the sole H3K4 methyltransferase and is mainly enriched at the promoters of actively transcribed genes. In contrast, Clr4 methyltransferase initiates H3K9 methylation, which has long been regarded as a hallmark of heterochromatic silencing. Lsd1 and Lsd2 are two highly conserved H3K4 and H3K9 demethylases. As these histone-modifying enzymes perform critical roles in maintaining histone methylation patterns and, consequently, gene expression profiles, cross-regulations among these enzymes are part of the complex regulatory networks. Thus, elucidating the mechanisms that govern their signaling and mutual regulations remains crucial. Here, we demonstrated that C-terminal truncation mutants, lsd1-ΔHMG and lsd2-ΔC, do not compromise the integrity of the Lsd1/2 complex but impair their chromatin-binding capacity at the promoter region of target genomic loci. We identified protein-protein interactions between Lsd1/2 and Raf2 or Swd2, which are the subunits of the Clr4 complex (CLRC) and Set1-associated complex (COMPASS), respectively. We showed that Clr4 and Set1 modulate the protein levels of Lsd1 and Lsd2 in opposite ways through the ubiquitin-proteasome-dependent pathway. During heat stress, the protein levels of Lsd1 and Lsd2 are upregulated in a Set1-dependent manner. The increase in protein levels is crucial for differential gene expression under stress conditions. Together, our results support a cross-regulatory model by which Set1 and Clr4 methyltransferases control the protein levels of Lsd1/2 demethylases to shape the dynamic chromatin landscape.

In situ visualization of m6A sites in cellular mRNAs.

N 6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is spatially regulated remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m6A sites in target RNAs which enables simultaneous detection of both m6A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m6A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m6A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m6A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m6A-modified transcript dynamics and investigating methylated RNA localization in individual cells.

Sphingobacterium phlebotomi sp. nov., a new member of family Sphingobacteriaceae isolated from sand fly rearing substrate.

A Gram-stain-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium, designated type strain SSI9T, was isolated from sand fly (Phlebotomus papatasi Scopoli; Diptera: Psychodidae) rearing substrate and subjected to polyphasic taxonomic analysis. Strain SSI9T contained phosphatidylethanolamine as a major polar lipid, MK-7 as the predominant quinone, and C16 : 1ω6c/C16 : 1ω7c, iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 0 as the major cellular fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that SSI9T represents a member of the genus Sphingobacterium, of the family Sphingobacteriaceae sharing 96.5-88.0 % sequence similarity with other species of the genus Sphingobacterium. The results of multilocus sequence analysis using the concatenated sequences of the housekeeping genes recA, rplC and groL indicated that SSI9T formed a separate branch in the genus Sphingobacterium. The genome of SSI9T is 5 197 142 bp with a DNA G+C content of 41.8 mol% and encodes 4395 predicted coding sequences, 49 tRNAs, and three complete rRNAs and two partial rRNAs. SSI9T could be distinguished from other species of the genus Sphingobacterium with validly published names by several phenotypic, chemotaxonomic and genomic characteristics. On the basis of the results of this polyphasic taxonomic analysis, the bacterial isolate represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium phlebotomi sp. nov. is proposed. The type strain is SSI9T (=ATCC TSD-210T=LMG 31664T=NRRL B-65603T).

The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes.

Attraction and oviposition preferences of Phlebotomus papatasi (Diptera: Psychodidae), vector of Old-World cutaneous leishmaniasis, to larval rearing media.

BACKGROUND: As part of a project aimed at developing oviposition attractants for the control and surveillance of Phlebotomus papatasi (a vector of Old-World cutaneous leishmaniasis), we tested the hypothesis that gravid sand flies are attracted to chemical cues emanating from the growth medium of conspecific larvae - predominantly larvae-conditioned host feces that represents a suitable oviposition site. We report the results of a systematic assessment of media from various developmental stages of the sand fly using oviposition and olfactometer behavioral assays. METHODS: We conducted multiple-choice oviposition assays in 500 mL Nalgene jars. Six treatments were placed on separate filter paper discs at the bottom of the jar: 2(nd)/3(rd) larval instar medium, 4(th) larval instar/pupae medium, frass from expired colonies, larval food (aged rabbit chow and rabbit feces mix), rabbit feces, and a solvent (water) control. Fifty gravid females were introduced into each jar. Cumulative number of eggs laid on each filter paper per jar was counted at different time intervals from digital images. Attraction of gravid sand flies to these six treatments was assayed with a 3-chamber linear olfactometer. Twenty gravid females were transferred to the middle chamber of the olfactometer and their distribution in treatment and control chambers was recorded after 3 h. RESULTS: Almost no eggs were oviposited during the first 72 h following a blood-meal. Cumulative egg deposition increased drastically in the next 24 h (hours 73-96), with a slight non-significant increasing trend thereafter. Comparing mean cumulative egg deposition among the six treatments, we found that significantly more eggs were oviposited on 2(nd)/3(rd) larval rearing medium followed by 4(th) instar/pupae rearing medium. Oviposition preference did not vary over time. The olfactometer results were consistent with the oviposition assays, with 2(nd)/3(rd) larval rearing medium being the most attractive, followed by 4(th) instar/pupae rearing medium. CONCLUSION: The key finding of this study is that gravid, laboratory reared, Ph. papatasi sand flies are significantly more attracted to rearing medium of the most biologically active larval stages (2(nd)/3(rd) instar and 4(th) instar/pupae). This finding indicates that sand fly-digested host food and feces is attractive to gravid females and suggests that the larvae and larval gut microbiome may be involved in conditioning the oviposition substrate and possibly the production of oviposition attractants and stimulants.