DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathway.

DIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3'end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.

Human Satellite 1A analysis provides evidence of pericentromeric transcription.

BACKGROUND: Pericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Human classical satellite DNAs are classified into three families named HSat1, HSat2, and HSat3, which have historically posed a challenge for the assembly of the human reference genome where they are misrepresented due to their repetitive nature. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSat1A has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of functional knowledge. Here, we aim to characterize and provide an understanding on the biological relevance of HSat1A. RESULTS: The path followed herein trails with HSat1A isolation and cloning, followed by in silico analysis. Monomer copy number and expression data was obtained in a wide variety of human cell lines, with greatly varying profiles in tumoral/non-tumoral samples. HSat1A was mapped in human chromosomes and applied in in situ transcriptional assays. Additionally, it was possible to observe the nuclear organization of HSat1A transcripts and further characterize them by 3' RACE-Seq. Size-varying polyadenylated HSat1A transcripts were detected, which possibly accounts for the intricate regulation of alternative polyadenylation. CONCLUSION: As far as we know, this work pioneers HSat1A transcription studies. With the emergence of new human genome assemblies, acrocentric pericentromeres are becoming relevant characters in disease and other biological contexts. HSat1A sequences and associated noncoding RNAs will most certainly prove significant in the future of HSat research.

Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration.

BACKGROUND: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. RESULTS: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. CONCLUSIONS: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Anthracyclines induce DNA damage response-mediated protection against severe sepsis.

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

IsomiR_Window: a system for analyzing small-RNA-seq data in an integrative and user-friendly manner.

BACKGROUND: IsomiRs are miRNA variants that vary in length and/or sequence when compared to their canonical forms. These variants display differences in length and/or sequence, including additions or deletions of one or more nucleotides (nts) at the 5' and/or 3' end, internal editings or untemplated 3' end additions. Most available tools for small RNA-seq data analysis do not allow the identification of isomiRs and often require advanced knowledge of bioinformatics. To overcome this, we have developed IsomiR Window, a platform that supports the systematic identification, quantification and functional exploration of isomiR expression in small RNA-seq datasets, accessible to users with no computational skills. METHODS: IsomiR Window enables the discovery of isomiRs and identification of all annotated non-coding RNAs in RNA-seq datasets from animals and plants. It comprises two main components: the IsomiR Window pipeline for data processing; and the IsomiR Window Browser interface. It integrates over ten third-party softwares for the analysis of small-RNA-seq data and holds a new algorithm that allows the detection of all possible types of isomiRs. These include 3' and 5'end isomiRs, 3' end tailings, isomiRs with single nucleotide polymorphisms (SNPs) or potential RNA editings, as well as all possible fuzzy combinations. IsomiR Window includes all required databases for analysis and annotation, and is freely distributed as a Linux virtual machine, including all required software. RESULTS: IsomiR Window processes several datasets in an automated manner, without restrictions of input file size. It generates high quality interactive figures and tables which can be exported into different formats. The performance of isomiR detection and quantification was assessed using simulated small-RNA-seq data. For correctly mapped reads, it identified different types of isomiRs with high confidence and 100% accuracy. The analysis of a small RNA-seq data from Basal Cell Carcinomas (BCCs) using isomiR Window confirmed that miR-183-5p is up-regulated in Nodular BCCs, but revealed that this effect was predominantly due to a novel 5'end variant. This variant displays a different seed region motif and 1756 isoform-exclusive mRNA targets that are significantly associated with disease pathways, underscoring the biological relevance of isomiR-focused analysis. IsomiR Window is available at https://isomir.fc.ul.pt/ .

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Genomic Tackling of Human Satellite DNA: Breaking Barriers through Time.

(Peri)centromeric repetitive sequences and, more specifically, satellite DNA (satDNA) sequences, constitute a major human genomic component. SatDNA sequences can vary on a large number of features, including nucleotide composition, complexity, and abundance. Several satDNA families have been identified and characterized in the human genome through time, albeit at different speeds. Human satDNA families present a high degree of sub-variability, leading to the definition of various subfamilies with different organization and clustered localization. Evolution of satDNA analysis has enabled the progressive characterization of satDNA features. Despite recent advances in the sequencing of centromeric arrays, comprehensive genomic studies to assess their variability are still required to provide accurate and proportional representation of satDNA (peri)centromeric/acrocentric short arm sequences. Approaches combining multiple techniques have been successfully applied and seem to be the path to follow for generating integrated knowledge in the promising field of human satDNA biology.

Molecular cytogenetic analyses reveal extensive chromosomal rearrangements and novel B chromosomes in Moenkhausia (Teleostei, Characidae).

The cytogenetic characteristics of five fish species of the Moenkhausia are described, based on the analysis of specimens collected in different headwater. All the species analyzed presented 2n=50 chromosomes. The C-banding revealed a similar distribution pattern of heterochromatic blocks in all the species, except Moenkhausia nigromarginata. The 5S rDNA sites were distributed on multiple chromosome pairs in all five species. Single and multiple histone H1 sites were observed in all the species, and histone H1 was shown to be co-located with the 18S rRNA gene in a single chromosome pair. The U2 snDNA gene was distributed at multiple sites in all the Moenkhausia species. The presence of B microchromosomes was confirmed in Moenkhausia forestii, while individuals of the three study populations of Moenkhausia oligolepis presented three morphologically distinct types of B chromosome. The chromosomal mapping of the 18S rDNA sites using the FISH technique revealed signals in the B chromosomes of M. forestii, while clusters of the H1 histone and U2 snDNA genes were found in the B chromosomes of M. forestii and M. oligolepis. The classical and molecular cytogenetic markers used in this study revealed ample variation in the Moenkhausia karyotypes, reflecting the dynamic nature of the chromosomal evolution.

A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells.

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Networks of mRNA Processing and Alternative Splicing Regulation in Health and Disease.

mRNA processing events introduce an intricate layer of complexity into gene expression processes, supporting a tremendous level of diversification of the genome's coding and regulatory potential, particularly in vertebrate species. The recent development of massive parallel sequencing methods and their adaptation to the identification and quantification of different RNA species and the dynamics of mRNA metabolism and processing has generated an unprecedented view over the regulatory networks that are established at this level, which contribute to sustain developmental, tissue specific or disease specific gene expression programs. In this chapter, we provide an overview of the recent evolution of transcriptome profiling methods and the surprising insights that have emerged in recent years regarding distinct mRNA processing events - from the 5' end to the 3' end of the molecule.

Linking amyotrophic lateral sclerosis and spinal muscular atrophy through RNA-transcriptome homeostasis: a genomics perspective.

In this review, we present our most recent understanding of key biomolecular processes that underlie two motor neuron degenerative disorders, amyotrophic lateral sclerosis, and spinal muscular atrophy. We focus on the role of four multifunctional proteins involved in RNA metabolism (TDP-43, FUS, SMN, and Senataxin) that play a causal role in these diseases. Recent results have led to a novel scenario of intricate connections between these four proteins, bringing transcriptome homeostasis into the spotlight as a common theme in motor neuron degeneration. We review reported functional and physical interactions between these four proteins, highlighting their common association with nuclear bodies and small nuclear ribonucleoprotein particle biogenesis and function. We discuss how these interactions are turning out to be particularly relevant for the control of transcription and chromatin homeostasis, including the recent identification of an association between SMN and Senataxin required to ensure the resolution of DNA-RNA hybrid formation and proper termination by RNA polymerase II. These connections strongly support the existence of common pathways underlying the spinal muscular atrophy and amyotrophic lateral sclerosis phenotype. We also discuss the potential of genome-wide expression profiling, in particular RNA sequencing derived data, to contribute to unravelling the underlying mechanisms. We provide a review of publicly available datasets that have addressed both diseases using these approaches, and highlight the value of investing in cross-disease studies to promote our understanding of the pathways leading to neurodegeneration.

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells.

The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3ß, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3ß or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.

The effects of nature-based vs. indoor settings on the adaptability, performance and affect of calisthenics exercisers. A registered report.

Comparisons of the beneficial effects of nature-based versus indoor physical activity have been extensively reported, but existing research addresses mainly aerobic activity (running, jogging), not resistance or mixed (aerobic and resistance) exercise. It is unclear if the psychological benefits extend to functionality, i.e., if participants perform their activities better in nature, and how movement is expressed in nature-based and indoor environments, during similar exercise. The present registered report was a randomized controlled trial investigating how engaging in similar resistance-based exercise (calisthenics) in nature-based and indoor settings differed in affective valence, perceived exertion, visual attention, movement adaptability, heart rate variability, and performance. Nature-based exercisers (N = 51) showed increased performance output than indoor exercisers (N = 53) (p < 0.001). There were no group differences in affective valence, perceived exertion, or visual attention. However, psychological states of nature-based exercisers showed stronger associations to performance output (r < 0.33) than those of indoor exercisers (r < 0.03). Nature-based exercisers' movement variability and structure, measured with non-linear and fractal techniques (Sample Entropy and Detrended Fluctuation Analysis), were more regular (p < 0.001) and more functionally adaptive (long-term Detrended Fluctuation Analysis, p = 0.022) to achieve better performance output. Heart rate variability measures were not different between groups. Distinct environments can influence movement adaptability in a calisthenics exercise routine, and ultimately contribute to better performance. These results show how action is specific to task environment, and how action implies not only the task, but also the characteristics of the environment. TRIAL REGISTRATION: NCT05090501 (Clinicaltrials.gov). Registered October 21, 2021.

Pericentromeric satellite RNAs as flexible protein partners in the regulation of nuclear structure.

Pericentromeric heterochromatin is mainly composed of satellite DNA sequences. Although being historically associated with transcriptional repression, some pericentromeric satellite DNA sequences are transcribed. The transcription events of pericentromeric satellite sequences occur in highly flexible biological contexts. Hence, the apparent randomness of pericentromeric satellite transcription incites the discussion about the attribution of biological functions. However, pericentromeric satellite RNAs have clear roles in the organization of nuclear structure. Silencing pericentromeric heterochromatin depends on pericentromeric satellite RNAs, that, in a feedback mechanism, contribute to the repression of pericentromeric heterochromatin. Moreover, pericentromeric satellite RNAs can also act as scaffolding molecules in condensate subnuclear structures (e.g., nuclear stress bodies). Since the formation/dissociation of nuclear condensates provides cell adaptability, pericentromeric satellite RNAs can be an epigenetic platform for regulating (sub)nuclear structure. We review current knowledge about pericentromeric satellite RNAs that, irrespective of the meaning of biological function, should be functionally addressed in regular and disease settings. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA in Disease and Development > RNA in Disease.

Hemophagocytic lymphohistiocytosis and macrophage activation syndrome: two rare sides of the same devastating coin.

Hemophagocytic lymphohistiocytosis (HLH) is a rare genetic hyperinflammatory syndrome that occurs early in life. Macrophage activation syndrome (MAS) usually refers to a secondary form of HLH associated with autoimmunity, although there are other causes of secondary HLH, such as infections and malignancy. In this article, we reviewed the concepts, epidemiology, clinical and laboratory features, diagnosis, differential diagnosis, prognosis, and treatment of HLH and MAS. We also reviewed the presence of MAS in the most common autoimmune diseases that affect children. Both are severe diseases that require prompt diagnosis and treatment to avoid morbidity and mortality.

Rare diseases: What rheumatologists need to know?

Although the terms "rare diseases" (RD) and "orphan diseases" (OD) are often used interchangeably, specific nuances in definitions should be noted to avoid misconception. RD are characterized by a low prevalence within the population, whereas OD are those inadequately recognized or even neglected by the medical community and drug companies. Despite their rarity, as our ability on discovering novel clinical phenotypes and improving diagnostic tools expand, RD will continue posing a real challenge for rheumatologists. Over the last decade, there has been a growing interest on elucidating mechanisms of rare autoimmune and autoinflammatory rheumatic diseases, allowing a better understanding of the role played by immune dysregulation on granulomatous, histiocytic, and hypereosinophilic disorders, just to name a few. This initiative enabled the rise of innovative targeted therapies for rheumatic RD. In this review, we explore the state-of-the art of rare RD and the critical role played by rheumatologists in healthcare. We also describe the challenges rheumatologists may face in the coming decades.

Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue.

Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

A cross-sectional survey of pain in palliative care in Portugal.

OBJECTIVE: The purpose of this work is to study the prevalence, intensity, and treatment of pain in Portuguese palliative care teams. METHODS: Twenty-one palliative care teams were invited to participate in a cross-sectional survey. Ten of these accepted and were included in the study. Data of all patients observed on the 18th week of 2011 were collected. The data collected concerning pain were: demographic data, pain intensity, drugs prescribed, and invasive techniques. The intensity of pain was rated using a five-point verbal rating scale from none to maximum. The Pain Management Index (PMI) was used to calculate the adequacy of the analgesia. RESULTS: A total of 164 patients were included in this study. One hundred fifty-one (92 %) had cancer. The median age was 71 years (16 to 95). Eighty-four (51 %) were females. Pain was directly assessed in 136 (83 %) of the patients, whereas 27 patients could not report pain because of cognitive failure. Of those directly assessed, 77 (57 %) had pain when they were assessed: 42 (55 %) mild, 25 (32 %) moderate, 9 (12 %) severe, and 1 (1 %) maximum. Non-opioid analgesics were used: paracetamol in 61 (37 %) and NSAID in 20 (12 %). Tramadol was the only opioid for mild to moderate pain used in 25 (15 %) patients. The opioids most used for moderate to intense pain were: morphine 74 (45 %), transdermal (TD) fentanyl 32 (20 %), and buprenorphine TD 28 (17 %). The adjuvants most used were: corticosteroids 38 (23 %), gabapentin 37 (23 %), and amitriptyline 15 (9 %). Only five (4 %) patients had a negative PMI, meaning an inadequate analgesia. CONCLUSION: The general prevalence of pain is similar to that reported by other. The prevalence of moderate to severe pain is also similar to that reported in other studies, although severe pain is somewhat lower than indicated in most reports. According to the PMI, pain control was acceptable to good.