Poly(A)-Specific Ribonuclease Mediates 3′-End Trimming of Argonaute2-Cleaved Precursor MicroRNAs

SummaryMicroRNAs (miRNAs) are typically generated as ∼22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3′ arm of the pre-miR-451 precursor hairpin by Ago2. The 3′ end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A)-specific ribonuclease (PARN) as the enzyme responsible for the 3′–5′ exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage

Serum amyloid alpha 1-2 are not required for liver inflammation in the 4T1 murine breast cancer model

がんに起因して起こる宿主の肝臓の急性期応答と炎症 --血清アミロイドαは乳がんモデルにおける肝臓の炎症の原因ではない--. 京都大学プレスリリース. 2023-02-06.Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause inflammation in various host organs. Despite the well-known coincidence of acute phase response and inflammation, the direct roles of SAA proteins in inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in liver inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. Consistently, 4T1-induced gene expression changes in bone marrow do not require Saa1-2. This study clarifies the negligible contribution of SAA1-2 proteins in liver inflammation in the 4T1 breast cancer model

Preterm toddlers have low nighttime sleep quality and high daytime activity.

A number of studies have been made on the sleep characteristics of children born preterm in an attempt to develop methods to address the sleep problems commonly observed among such children. However, the reported sleep characteristics from these studies vary depending on the observation methods used, i.e., actigraphy, polysomnography and questionnaire. In the current study, to obtain reliable data on the sleep characteristics of preterm-born children, we investigated the difference in sleep properties between 97 preterm and 97 term toddlers of approximately 1.5 years of age using actigraphy. Actigraphy units were attached to the toddlers’ waists with an adjustable elastic belt for 7 consecutive days, and a child sleep diary was completed by their parents. In the study, we found that preterm toddlers had more nocturnal awakenings and more daytime activity, suggesting that preterm-born children may have a different process of sleep development in their early development

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast

International audienceAntisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity

Remote solid cancers rewire hepatic nitrogen metabolism via host nicotinamide-N-methyltransferase

がんによって全身に不調が生じるのはなぜか? --がんをもつ個体の肝臓の異常に焦点をあてる--. 京都大学プレスリリース. 2022-06-16.Cancers disrupt host homeostasis in various manners but the identity of host factors underlying such disruption remains largely unknown. Here we show that nicotinamide-N-methyltransferase (NNMT) is a host factor that mediates metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid cancers distantly increase expression of Nnmt and its product 1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal suppression of the urea cycle accompanied by accumulation of amino acids, and enhancement of uracil biogenesis in the livers of cancer-bearing mice. Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic abnormalities, and buffers cancer-dependent weight loss and reduction of the voluntary wheel-running activity. Our data also demonstrate that MNAM is capable of affecting urea cycle metabolites in the liver. These results suggest that cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby disrupting host homeostasis

Bases of antisense lncRNA-associated regulation of gene expression in fission yeast

<div><p>Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene <i>ctt1</i> revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to <i>ctt1</i> induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on <i>ctt1</i> induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.</p></div

Dicer controls <i>ctt1</i> induction independently of Exo2.

<p><b>A.</b> Analysis of <i>ctt1</i> mRNA induction in WT, <i>exo2</i>Δ, <i>dcr1</i>Δ and <i>exo2</i>Δ <i>dcr1</i>Δ cells. Strains YAM2400 (WT), YAM2402 (<i>exo2</i>Δ), YAM2406 (<i>dcr1</i>Δ) and YAM2404 (<i>exo2</i>Δ <i>dcr1</i>Δ) were grown as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. <i>ctt1</i> mRNA was quantified by strand-specific RT-qPCR as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2C</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001 upon t-test. <b>B.</b> Analysis of <i>XUT0794</i> level in in WT, <i>exo2</i>Δ, <i>dcr1</i>Δ and <i>exo2</i>Δ <i>dcr1</i>Δ cells. Strains YAM2400 (WT), YAM2402 (<i>exo2</i>Δ), YAM2406 (<i>dcr1</i>Δ) and YAM2404 (<i>exo2</i>Δ <i>dcr1</i>Δ) were grown to mid-log phase in rich medium. <i>XUT0794</i> was quantified by strand-specific RT-qPCR as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2C</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; ns, not significant upon t-test. <b>C.</b> ChIP analysis of RNAPII occupancy across <i>ctt1</i> in <i>dcr1</i>Δ cells. Strains YAM2400 (WT) and YAM2406 (<i>dcr1</i>Δ) were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. Cross-linking, chromatin extraction and data analysis were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g004" target="_blank">Fig 4B</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. ns, not significant upon t-test. <b>D.</b> ChIP analysis of H4K5/8/12/16 acetylation (H4-acetyl) along <i>ctt1</i> in <i>dcr1</i>Δ cells. Strains YAM2400 (WT) and YAM2406 (<i>dcr1</i>Δ) were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. Cross-linking and chromatin extraction were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g004" target="_blank">Fig 4B</a>. Data analysis was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g005" target="_blank">Fig 5C</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. *<i>p</i><0.05; **<i>p</i><0.01 upon t-test.</p

Genome-wide identification of class 1 genes in <i>S</i>. <i>pombe</i>.

<p><b>A.</b> Transcriptional attenuation in <i>exo2</i>Δ cells. NET-Seq analysis was performed from biological duplicates of WT and <i>exo2</i>Δ cells. Data for the WT strain were previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.ref030" target="_blank">30</a>]. After sequencing, differential analysis discriminated genes showing significant (<i>P</i><0.05) reduction of transcription (classes 1–2) or not (classes 3–4). Among them, classes 1 and 3 have asXUT. The number of genes for each class is indicated. <b>B.</b> Box-plot of nascent transcription (NET-Seq) signal for class 1–4 genes in WT (dark grey boxes) and <i>exo2</i>Δ (Δ; light grey boxes). <b>C.</b> Metagene view of NET-Seq signals along class 1 and 3 genes in WT cells. For each class, normalized coverage (tag/nt, log₂) along mRNA transcription start site (TSS) +/- 1000 nt (sense) and the antisense (as) strand were piled up, in a strand-specific manner. Average signal for the sense and antisense strands was plotted for class 1 (red) and class 3 (green). The shading surrounding each line denotes the 95% confidence interval. <b>D.</b> Same as above in <i>exo2</i>Δ. <b>E.</b> Density-plot showing the global NET-Seq (dashed lines) and total RNA-Seq (solid lines) signals for XUTs in the WT (black) and <i>exo2</i>Δ (pink) strains. Total RNA-Seq data were previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.ref030" target="_blank">30</a>]. <b>F.</b> Metagene view of H4K5/8/12/16 acetylation (H4ac) for class 1 (red), class 2 (blue), class 3 (green) and class 4 (black) genes in WT cells. ChIP-Seq libraries construction and sequencing were previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.ref030" target="_blank">30</a>]. Metagene representation of signal for each class of genes was performed as above, in a strand-unspecific manner, using ratio of coverage (log₂) for H4ac and H3. The shading surrounding each line denotes the 95% confidence interval.</p

Role of Set2-dependent H3K36me3 in <i>ctt1</i> attenuation.

<p><b>A.</b> Set2 is recruited to <i>ctt1</i> upon oxidative stress. YAM2816 (Set2-13Myc [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.ref060" target="_blank">60</a>]) cells were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. Cross-linking and chromatin extraction were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g004" target="_blank">Fig 4</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. <b>B.</b> ChIP analysis of H3K36 trimethylation (me3) along <i>ctt1</i> in <i>exo2</i>Δ cells. Strains YAM2400 (WT) and YAM2402 (<i>exo2</i>Δ) were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. Cross-linking and chromatin extraction were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g004" target="_blank">Fig 4</a>. Data analysis was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g005" target="_blank">Fig 5C</a>. Average values and SEM were calculated from three biological replicates. *<i>p</i><0.05; ns, not significant upon t-test. <b>C.</b> Analysis of <i>XUT0794</i> level in <i>set2</i>Δ cells. Strains YAM2400 (WT), YAM2402 (<i>exo2</i>Δ) and YAM2797 (<i>set2</i>Δ) were grown to mid-log phase in rich medium. <i>XUT0794</i> level was quantified from total RNA as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2C</a>. Data are presented as mean +/- SEM, calculated from three biological replicates. ** <i>p</i> < 0.01; *** <i>p</i> < 0.001; ns, not significant upon t-test. <b>D.</b> Analysis of <i>ctt1</i> mRNA induction in <i>set2</i>Δ cells. Strains YAM2400 (WT), YAM2402 (<i>exo2</i>Δ) and YAM2797 (<i>set2</i>Δ) were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2B</a>. <i>ctt1</i> mRNA was quantified by strand-specific RT-qPCR as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007465#pgen.1007465.g002" target="_blank">Fig 2C</a>. Data are presented as above. *** <i>p</i> < 0.001; ns, not significant upon t-test.</p