Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.
- MeSH
- DEAD-box RNA-helikasy genetika metabolismus MeSH
- dvouvláknová RNA * metabolismus genetika MeSH
- malá interferující RNA * genetika metabolismus MeSH
- mikro RNA genetika metabolismus MeSH
- myši MeSH
- protein - isoformy genetika metabolismus MeSH
- ribonukleasa III * genetika metabolismus MeSH
- RNA interference * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The profile of the antitumor immune response is an important factor determining patient clinical outcome. However, the influence of the tissue contexture on the composition of the tumor microenvironments of virally induced tumors is not clearly understood. Therefore, we analyzed the immune landscape of two HPV-associated malignancies: oropharyngeal squamous cell carcinoma (OPSCC) and squamous cell carcinoma of uterine cervix (CESC). We employed multiplex immunohistochemistry and immunofluorescence to evaluate the density and spatial distribution of immune cells in retrospective cohorts of OPSCC and CESC patients. This approach was complemented by transcriptomic analysis of purified primary tumor cells and in silico analysis of publicly available RNA sequencing data. Transcriptomic analysis showed similar immune profiles in OPSCC and CESC samples. Interestingly, immunostaining of OPSCC tissues revealed high densities of immune cells in both tumor stroma and tumor epithelium, whereas CESC samples were mainly characterized by the lack of immune cells in the tumor epithelium. However, in contrast to other immune cell populations, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were abundant in both segments of CESC samples and CESC-derived tumor cells expressed markedly higher levels of the PMN-MDSC chemoattractants CXCL1, CXCL5, and CXCL6 than OPSCC tumor cells. Taken together, despite their having the same etiologic agent, the immune infiltration pattern significantly differs between OPSCC and CESC, with a noticeable shift toward prominent MDSC infiltration in the latter. Our data thus present a rationale for a diverse approach to targeted therapy in patients with HPV-associated tumors of different tissue origins.
- Publikační typ
- časopisecké články MeSH
Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
- MeSH
- mikro RNA * genetika metabolismus MeSH
- myši MeSH
- ribonukleasa III * metabolismus MeSH
- RNA interference MeSH
- savci metabolismus MeSH
- transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
Vydání první 12 nečíslovaných stran : ilustrace (některé barevné) ; 30 cm
Naučný komiks pro děti, který se zaměřuje na dějiny genetiky a na genom a DNA.
- Konspekt
- Obecná genetika. Obecná cytogenetika. Evoluce
- Literatura pro děti a mládež (naučná)
- NLK Obory
- genetika, lékařská genetika
- dějiny lékařství
- NLK Publikační typ
- komiksy
- literatura pro děti a mládež
First edition 12 nečíslovaných stran : ilustrace (některé barevné) ; 30 cm
A comic book for children, that teaches about the history of genetics and about the genome and the DNA.
- Konspekt
- Obecná genetika. Obecná cytogenetika. Evoluce
- Literatura pro děti a mládež (naučná)
- NLK Obory
- genetika, lékařská genetika
- dějiny lékařství
- NLK Publikační typ
- komiksy
- literatura pro děti a mládež
Plasmacytoid dendritic cells (pDCs) are the most potent type I interferon-producing cells and play an important role in antiviral immunity. Tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce interferon alpha (IFNα) and confirmed capacity to prime regulatory T cells (Tregs) by the ICOS/ICOS-L pathway. Because a significant number of HNSCCs are induced by human papillomaviruses and show markedly different immune profiles than non-virally induced tumors, we compared the phenotype and functional capacity of HNSCC-infiltrating pDCs to the HPV status of the tumor. We observed a reduced capacity of pDCs to produce IFNα upon toll-like receptor activation in HPV-negative samples and a rather uncompromised functionality in HPV-associated tumors. Additionally, supernatants from non-virally induced but not HPV-associated tumor cell suspensions significantly inhibited IFNα production by peripheral blood-derived pDCs. We identified IL-10 and TNFα as the soluble pDC-suppressive factors with the highest variability between HPV-negative and HPV-positive tumor-derived supernatants. Additionally, we observed a positive correlation of tumor-infiltrating pDCs with Tregs in HPV-negative samples but not in virally induced tumors. Overall, our study indicates that the immunosuppressive cytokine milieu rich in IL-10 and TNFα in HPV-negative but not in HPV-positive HNSCC significantly affects the functional capacity of tumor-infiltrating pDCs, and such dysfunctional pDCs may further support the immunosuppressive tumor microenvironment by promoting the expansion of Tregs in the tumor tissue.
- MeSH
- biologické markery MeSH
- cytokiny metabolismus MeSH
- dendritické buňky imunologie metabolismus patologie MeSH
- dlaždicobuněčné karcinomy hlavy a krku etiologie metabolismus patologie MeSH
- exprese genu MeSH
- infekce papilomavirem komplikace virologie MeSH
- interferon alfa imunologie metabolismus MeSH
- lidé MeSH
- náchylnost k nemoci MeSH
- nádorové mikroprostředí * imunologie MeSH
- regulační T-lymfocyty imunologie metabolismus MeSH
- studie případů a kontrol MeSH
- T-lymfocyty - podskupiny imunologie metabolismus MeSH
- virová transformace buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Germline genome defense evolves to recognize and suppress retrotransposons. One of defensive mechanisms is the PIWI-associated RNA (piRNA) pathway, which employs small RNAs for sequence-specific repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes posses also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway, we produced a new RNAi pathway mutant DicerSOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA gene. Normal follicular and oocyte development in double mutants showed that RNAi does not suppress a strong ovarian piRNA knock-out phenotype. However, we observed redundant and non-redundant targeting of specific retrotransposon families illustrating stochasticity of recognition and targeting of invading retrotransposons. Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MaLR and RLTR10 retrotransposons were targeted mainly by RNAi. Double mutants showed accumulations of LINE-1 retrotransposon transcripts. However, we did not find strong evidence for transcriptional activation and mobilization of retrotransposition competent LINE-1 elements suggesting that while both defense pathways are simultaneously expendable for ovarian oocyte development, yet another transcriptional silencing mechanism prevents mobilization of LINE-1 elements.
- MeSH
- Argonaut proteiny genetika MeSH
- DEAD-box RNA-helikasy genetika MeSH
- malá interferující RNA genetika MeSH
- mutace MeSH
- myši MeSH
- oocyty chemie růst a vývoj MeSH
- retroelementy * MeSH
- ribonukleasa III genetika MeSH
- RNA interference * MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
