Ribonucleotide Protein Research Articles

Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.

Abstract 7480: Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer

Abstract The aberrant expression of repeat elements is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). The majority of these are non-coding RNAs that have shown the ability to stimulate a viral-like innate immune response. The long interspersed nuclear element 1 (LINE-1) retrotransposon is one of the few protein coding repeat elements in the human genome. LINE-1 has two open reading frames, ORF1 and ORF2. The former (ORF1p) encodes a ribonucleotide protein and the latter (ORF2p) a reverse transcriptase and endonuclease. Although nuclear ORF1p and ORF2p is required for retrotransposition, ORF1p is primarily localized in the cytoplasm, which suggests the possibility of ORF1p function independent of LINE-1 retrotransposition. Here, we show that ORF1p has a function to shield repeat RNAs from activating pathogen recognition receptor (PRR) mediated antiviral response in PDAC cells. LINE-1 ORF1p-targeting shRNA significantly inhibits patient-derived PDAC tumor growth both in vitro and in vivo. Total RNA sequencing (RNA-seq) reveals distinct transcriptional profiles with ORF1p deficiency. Gene set enrichment analysis (GSEA) identified high enrichment of innate immune response-related pathways including IL6-JAK-STAT3 signaling, inflammatory response, interferon response, and apoptosis in ORF1 knockdown PDAC cell lines. These immune related pathways are suppressed by deletion of RIG-I and MAVS, two components of viral RNA sensing. Mechanistically, we demonstrated that ORF1p is associated with repeat RNAs including LINE-1 RNA (L1HS) in processing bodies (PBs), which are cytoplasmic membraneless ribonucleoprotein (RNP) granules. In the PBs, ORF1p interacts with MOV10 RNA helicase, an essential enzyme for dsRNA unwinding. Using MOV10 truncated mutants, we found that the ORF1p-MOV10 interaction is important in attenuation of PRR sensing and apoptosis upon repeat RNA exposure. Finally, we evalulated the activation of the innate immune response in the tumor microenvironment by ORF1p depletion. These results suggest that ORF1p loss induces a desmoplastic reaction driven by Acta2+ myofibroblastic CAFs in response to inflammatory cytokines. Collectively, our results demonstrate a critical and previously uncharacterized role of ORF1p in evasion of endogenous repeat RNA-triggered antiviral immune response in PDAC cells with implications in the tumor microenvironment. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Natalie Ho, Siyu Sun, Michael Raabe, Yuhui Song, Ildiko Phillips, Katherine Xu, Peter Richieri, Linda Nieman, Benjamin Greenbaum, David Ting. Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7480.

RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants.

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.

KDM6A Controls Genes Modulating Immune Surveillance in Multiple Myeloma

Background: In multiple myeloma (MM), inactivating mutations and loss of the histone demethylase KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) locus are found in up to 50% of patients and are associated with poorer prognosis. KDM6A belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex activates enhancers by adding activation marks on histones and removing methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. The H3K27me3 methyltransferase EZH2 that catalyzes the reverse reaction of KDM6A, is often overexpressed in MM and this also correlates with poor prognosis. As well, KMT2C, KMT2D and SWI/SNF components of its complex are all found significantly mutated in MM which emphasize the importance of enhancer deregulation in myeloma. Aim: Our goal is to understand how loss of KDM6A affects gene transcription networks and enhancer function to affect MM pathogenesis or progression. Method: We explored the transcriptional consequences of KDM6A deficiency in patient tumors using the gene expression dataset included within the Multiple Myeloma Research Foundation (MMRF) Researcher Gateway and in MM cell lines using data from the Cancer Cell Line Encyclopedia (CCLE). We further modeled the loss of KDM6A in female MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology and by re-expressing KDM6A in cells null for KDM6A. Using ChIP-sequencing analysis in isogenic KDM6A knockout or replete cell lines, we identified KDM6A binding site in MM and explored how KDM6A deficiency affects chromatin structure to regulate a transcriptional network. Results: Epithelial-to-mesenchymal-transition (EMT)-related pathways were downregulated in low KDM6A expressing tumor from the MMRF patient cohort. In both male and female patients, low expression of KDM6A was associated with decreased expression of genes involved in immune recognition. In MM cell lines, we observed a significant positive correlation between KDM6A expression level and expression of the transcriptional regulators of Major Histocompatibility complex I and II (MHCII and MCHII), NLRC5 and CIITA, respectively. With ChIP-sequencing analysis we find that KDM6A directly binds NLRC5 and CIITA genes, among many other genes involved in immune surveillance. KDM6A null MM cells displayed upregulation of pathways promoting EMT and downregulation of genes regulating immune function, such as HLA-A/B/C, CCL3/5, HHLA2 and ITGAL. Moreover, re-expression of KDM6A in KDM6A null cells upregulated the expression of these gene along with surface expression of MHCI proteins. Conclusion: Together, our data demonstrates that KDM6A modulates EMT and directly regulates expression of major regulators of immune surveillance. KDM6A deficiency may drive MM towards an EMT program to promote systemic spread and facilitate escape from the immune surveillance. Disclosures No relevant conflicts of interest to declare.

PP 6.8.16 - Cleaving HIV-1 provirus from ART-suppressed patient-derived resting CD4 T Cells using Cpf1/crRNA-array ribonucleotide protein packaged by CD4-targeting lentivirus-like particle

PP 6.8.16 - Cleaving HIV-1 provirus from ART-suppressed patient-derived resting CD4 T Cells using Cpf1/crRNA-array ribonucleotide protein packaged by CD4-targeting lentivirus-like particle

200 Developing exosomes as a mediator for CRISPR/Cas-9 delivery

CRISPR/Cas systems present a powerful gene-editing tool with the potential for widespread therapeutic use; however, current methods of in vivo delivery such as adeno-associated viruses (AAV) may stimulate an immune response, creating the need for an alternative for delivery of CRISPR/Cas9. Exosomes are small vesicles that are released by cells and serve as a delivery system for RNA, proteins, and various molecules to other cells. The focus of this project was to use exosomes as a delivery system for Cas9, exploiting their high uptake by target cells and their ability to avoid the immune system in vivo. Porcine fetal fibroblasts (PFF) were grown to 80% confluency; after 48h, exosomes were isolated and concentrated from conditioned media by filtration with a 0.22-μm filter followed by 100-kDa molecular weight cutoff filter. Transmission electron microscopy, Western blotting for presence of CD81, and an uptake assay for exosomes stained with the lipophilic dye DiI (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were used to characterise isolated exosomes, and average particle size was evaluated by NanoSight (Salisbury, United Kingdom). After characterisation, exosomes were loaded with Cas9 (PNA Bio, Newbury Park, CA, USA) using sonication, incubation with saponin, or extrusion. For each method of loading, 1.0×1011 exosomes and 500ng of Cas9 were used. For sonication, exosomes and Cas9 were sonicated 4 times: 4s on/2s off, left on ice for 2min, and then repeated for 4 more cycles. Loaded exosomes were then incubated at 37°C for 20min. For incubation with saponin, 100μL of 0.6% saponin solution was made in PBS, mixed with exosomes and Cas9, and then incubated on a shaker at 800 rpm for 20min. For extrusion, exosomes and Cas9 were extruded (Avanti Polar Lipids, Alabaster, AL, USA) 10, 15, or 20 times through a 0.22-μm filter. To evaluate efficiency of Cas9 loading into exosomes, loaded exosome samples were split in half, with one-half receiving a proteinase K digest (100μg mL−1) to remove free Cas9 and the other receiving no treatment. Proteinase K-treated and untreated samples were then compared side by side on Western blot staining for Cas9. ImageJ software (National Institutes for Health, Bethesda, MD, USA) was used to quantify band intensity and loading efficiency. With optimal conditions, our preliminary results show loading efficiency for sonication and saponin to be 16.7 and 19.2%, respectively, whereas loading by extrusion was undetectable. For CRISPR/Cas targeting, transgenic PFF carrying one copy of H2B-GFP were used to test delivery of ribonucleotide protein complex (RNP). To verify efficiency of the guide (g)RNA targeting green fluorescent protein (GFP), cells were nucleofected with Cas9 and gRNA. The DNA was extracted, PCR amplified, and sequenced (Eton Bioscience, San Diego, CA, USA) and then evaluated for indels with TIDE, resulting in a 53.2% cleavage efficiency. Next, exosomes will be loaded with RNP to knockout GFP in H2B-GFP cells, and targeting efficiency will be evaluated by flow cytometry and TIDE. We hypothesise that based on loading efficiency and target cell uptake, exosomes will present a safe and efficient method for in vitro and in vivo delivery of Cas9. The financial support of the Comparative Medicine Institute is gratefully acknowledged.

Modeling Human Paroxysmal Nocturnal Hemoglobinuria Via CRISPR/Cas9 HSPC Gene Editing in Non-Human Primate

Recent advances in CRISPR/Cas9 technology allowing precise genome editing at a site of interest have enabled relevant human disease modeling and the development of corrective gene therapies for various genetic disorders. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disorder linked to acquired somatic loss-of-function mutations disrupting the X-linked PIG-A gene in hematopoietic stem and progenitor cells (HSPC), characterized by the clinical triad of intravascular hemolysis, thrombosis and bone marrow failure, as well as clonal expansion of HSPC defective in glycosylphophatidylinositol(GPI)-linked proteins, due to loss of PIG-A enzymatic activity. There have been many attempts to model PNH utilizing conditional knockout strategies in mice, but these do not recapitulate the hemolytic phenotype or clonal dominance. Whether clonal expansion is intrinsic to PIG-A deficient HSPC or extrinsic, resulting from relative protection of PIG-A deficient HSPC and progeny cells from immune attack, is unclear. To explore the pathophysiologic mechanisms of PNH HSPC clonal expansion, we generated a relevant non-human primate model for PNH, utilizing autologous transplantation of HSPC edited with CRISPR/Cas9 at the PIG-A locus.The most efficient guide RNAs (gRNAs) targeting several sites within PIG-A exon 2 locus on the X chromosome, where most of mutations occur in patients with PNH, were selected based on efficiency of editing as screened in FRhK-4 cell lines and mobilized macaque CD34+ HSPC. Compared to editing of the control AAVS1 locus and other genes of interest, editing of all PIG-A locus sites was relatively inefficient. Autologous HSPC were transduced with ribonucleotide protein (RNP) complexes of Cas9 protein and selected gRNAs targeting either PIG-A or the AAVS1 “safe harbor” site, as an internal control, followed by transplantation into one female (ZI35) and one male (ZL19) macaque, respectively. Both animals engrafted promptly and clones acquiring the PNH loss-of-function phenotype, defined by loss of binding of FLAER, a fluorescent compound that binds to all GPI anchors, and loss of expression of lineage-specific GPI-linked proteins (CD24 for granulocytes and CD14 for monocytes) were stably maintained at levels of 0.2-0.4% for up to 19 months post-transplantation, with no evidence for intrinsic clonal expansion of PIG-A edited cells (Fig. 1A). Upon targeted deep sequencing, 0.4-1% insertions and deletions (INDELs) induced by CRISPR/Cas9 were detected, and the predominant INDEL type was identified as a single base deletion at the +1 or -1 positions of the target site consistently in granulocytes and other mature hematopoietic lineage cells from peripheral blood (PB), suggesting that initial mutations occurred in long-lasting HSPCs rather than short-term progenitor cells. Furthermore, as the PIG-A gene is located on X chromosome, we sought to investigate the difference in genome editing efficiency depending on the number of X alleles or activation state. Note that the expected INDEL frequency in completely PIG-A deficient sorted FLAERneg cells would be 100%, however, large deletions or rearrangements are not detected by standard deep sequencing methodologies. Interestingly, the mutation frequencies in total granulocytes and more importantly in sorted FLAER negative PNH cells were always much higher in male (ZL19) macaque cells than in female (ZI35) macaque cells (Fig. 1B and C). Consistently, gene editing PIG-A allele efficiency with CRISPR/Cas9 was also higher in human male B-lymphoblastoid cell lines (LCL) compared to female cells, 23.2% versus 16.6% (n=5), respectively. The finding that edited allele frequency was consistently lower in sorted FLAERneg female than male cells suggests that editing of the active X allele may be favored, potentially due to poor accessibility of inactive loci to editing machinery.In conclusion, we have successfully established a rhesus macaque model for PNH utilizing autologous transplantation of CRISPR/Cas9 edited HSPC. To date, we found no evidence for intrinsic expansion of PIG-A deficiency HSPC and hematopoietic progeny. This modeling approach could be utilized for further investigation of extrinsic or intrinsic factor responsible for clonal expansion. Furthermore, our findings provide a better insight into the relationship between CRISPR/Cas9 editing efficiency and active versus inactive X-linked genes. [Display omitted] DisclosuresDunbar:National Institute of Health: Research Funding.

Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM.We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM.Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. DisclosuresLicht:Celgene: Research Funding.

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex.

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.

Follicular Dendritic Cell Activation by TLR Ligands Promotes Autoreactive B Cell Responses

A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.

Abstract 5476: A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the United States, and the 3rd most common cancer associated with BRCA mutations. Frontline therapies have significant toxicities and only minimally extend overall survival, highlighting the need to optimize targeted therapies. Poly-ADP ribose polymerase (PARP) inhibitors (PARPi), a ‘poster child’ for personalized medicine, depend on the concept of synthetic lethality where the combined perturbation of DNA repair genes, via genetic mutations within the tumor cells, and pharmacological PARP inhibition effectively targets BRCA-deficient tumors. Although PARPi have delivered promising preclinical and clinical results, initially- responsive patients ultimately develop resistance. A unique mechanism elucidated by our lab demonstrates that the mRNA-binding protein HuR mediates resistance to DNA damaging agents through post-transcriptional regulation of select mRNA cargo. Predominantly expressed in the nucleus, HuR translocates to the cytoplasm upon tumor-associated stress. Cytoplasmic HuR binds and stabilizes unique pro-survival transcripts, resulting in resistance to a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi response. Methods and Results: Through immunofluorescence and western blot of fractionated lysates, we demonstrate that the PARP inhibitors Veliparib, Olaparib, and Rucaparib induced cytoplasmic HuR localization. Conversely, pre-treatment with MS-444 (Novartis), an established small molecule inhibitor of HuR, abrogated its nuclear export induced by PARPi treatment. Consistent with these findings, the growth-inhibitory effects of PARPi treatment were significantly potentiated upon HuR silencing whereas ectopic HuR overexpression promoted resistance, as observed in short term cell survival and long-term anchorage-independent growth assays. Additionally, silencing of HuR enhanced PARPi-induced cytotoxicity, assessed by increased accumulation of DNA damage (γH2Ax) foci and Poly ADP-ribose (PAR) polymers. Ribonucleotide protein immunoprecipitation (RNP-IP) assays demonstrated that HuR binds and upregulates Poly-ADP Ribose Glycohydrolase (PARG) mRNA, the major enzyme responsible for catabolism of PAR. Taken together, when PDA cells are exposed to PARPi, HuR mediates upregulation of PARG, thereby decreasing PARylation and facilitating DNA repair. Conversely, HuR inhibition results in detrimental accumulation of PAR and enhanced DNA damage, which ultimately leads to increased PARPi-conferred cytotoxicity. Discussion: These results demonstrate that HuR imposes a significant barrier to PARPi therapy by orchestrating a strong chemoresistance mechanism. Thus, we provide evidence that HuR (and/or its target) inhibition via an HuR inhibitor (MS-444) can optimize PARPi-based therapies for better patient outcomes. Citation Format: Saswati N. Chand, Akshay R. Kamath, Nicole Meisner-Kober, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2015-5476

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1–HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.

Intestinal Pseudo-obstruction: Initial Manifestation of Systemic Lupus Erythematosus

A 20-year-old man was hospitalized for 2 months after the onset of vomiting, cramping abdominal pain and dysuria. He had been diagnosed as IgA nephropathy 4 months ago. His mother was recently diagnosed as systemic lupus erythematosus (SLE). Physical examination revealed hypoactive bowel sounds and distended abdomen with diffuse tenderness. There was no rash on the face and body. The laboratory investigations showed hemoglobin 11.2 g/dL, leukocyte 10,500/µL, platelet 160,000/µL and albumin 3.2 g/dL. Antinuclear antibody (titer of 1:320 with speckled pattern), anti-Sm and ribonucleotide protein antibodies were positive. The 24 hour urine protein was 1,100 mg/day. Abdominal X-ray demonstrated multiple air-fluid levels of small bowel (Fig. 1). Abdominal CT showed fluid-filled dilatation of small bowel, bilateral hydronephrosis and thickened bladder wall. There was no abnormality on esophagogastroduodenoscopy and colonoscopy. Anorectal manometry showed decreased anal sphincter pressure and intact anaorectal inhibitory reflex. Figure 1 Simple abdominal erect view. Simple abdominal erect film shows extensive dilatation of the small bowel and multiple air-fluid levels. He was treated with empirical prednisolone 30 mg/day for 3 days but abdominal pain was worsened and plain X-ray showed aggravation of ileus. To find out the underlying cause of intestinal pseudo-obstruction (IPO), subtotal colectomy was performed. Grossly there was no mass, ulcer or mechanical obstruction. Histopathology demonstrated diffuse muscular degeneration with little vasculitis suggestive of SLE (Fig. 2). Steroid and azathiopurine were started after surgery and he became well. Figure 2 Microscopic findings of colon. (A) There is diffuse muscular degeneration, fibrosis and multifocal eosinophil infiltration. (B) The vascular lumen is intact and few inflammatory cells are infiltrated around the vessel. (C) Ganglinoic cells are increased ... After IPO in SLE was first described in 1993,1 few cases have been reported thereafter.2-4 As in this case, IPO may appear as the initial presentation of SLE2 and be associated with interstitial cystitis.3-5 The pathophysiology of IPO in SLE is dysmotility of the intestinal muscle, caused by neuropathy, vasculitis or primary myopathy.3-5 As IPO usually respond to high-dose steroid and additional immunosuppressant, early diagnosis and timely initiation of treatment are important to avoid unnecessary surgery.6

Distinctly different purpura on different arms

A 44-year-old woman, with severe lupus-associated renal failure needing haemodialysis, was admitted with purpura on both arms. Only the eruptions on her left arm were painful. Blood test results showed low platelets (6×109 per L), reduced complement activity, and high concentrations of antibodies to double-stranded DNA and ribonucleotide protein. Examination showed purpura aff ecting both arms, with a distinctly asymmetric appearance. Closer inspection of the right arm showed fl at, painless confl uent ecchymosis (fi gure A). By contrast, the left arm showed haemorrhagic vesicular (fi gure B) eruptions in a dermatomal pattern (fi gure C). Histological examination of a sample from one of the vesicles revealed multinucleated giant cells supportive of herpes zoster. Both the left-sided arm pain and haemorrhagic vesicles responded well to valaciclovir treatment. Skin bruising (ecchymosis) can result from several mechanisms, giving rise to distinct patterns and terminology. Purpura refers broadly to the spectrum of patterns and mechanistic processes that arise from extravasation of blood into skin. When purpuric eruptions are diminutive or pinpoint in size, they are termed petechiae. Purpura can result from dangerously low platelet concentrations (eg, idiopathic thrombocytopenic purpura), impaired platelet function (eg, eff ect of aspirin), congenital or acquired clotting disorders (eg, haemophilia or disseminated intravascular coagulation), or from an intense infl ammatory or structural injury to the blood vessel wall (eg, vasculitis vs scurvy). Various patterns include palpable purpura overlying the buttocks and legs (eg, Henoch-Schonlein purpura), involvement of palms and soles (eg, disseminated meningococcal or gonococcal infection), and a dermatomal distribution arising from concomitant herpes zoster infection and profound thrombocytopenia (fi gure C). Lancet 2009; 374: 1010

The role of lung resistance protein in radiation treated head and neck carcinoma

Objectives. Vault proteins are ribonucleotide proteins composed largely of a single large protein termed major vault protein. (MVP) The (MVP) LRP gene is located on chromosome 16 close to the genes coding for multidrug resistance‐associated protein and protein kinase c‐β. The role of MVP (LRP) in cancer drug resistance has been demonstrated in various cell lines as well as in ovarian carcinomas and acute myeloid leukaemia, however little is known about its possible role in radiation resistance.1,2 Our aim was to examine this in head and neck squamous carcinoma (HNSCC). Method. One hundred and thirty three patients with proven squamous cell carcinoma of the oropharynx were recruited into the study, looking at two subsites of posterior tongue and tonsil. These patients received primary radiotherapy with curative intent. Archived biopsy material was obtained for 78 patients. Immunohistochemistry was used to detect LRP expression. Locoregional failure and cancer specific survival were estimated using Kaplan Meier method. Results. On analysis of the whole group together LRP expression was strongly associated with locoregional failure (P < 0.0001) however, on subgroup analysis there was an obvious difference between the two sites, with increased LRP expression being associated with poor prognosis in the tongue group (P < 0.0001), but not the tonsil group. (P = 0.14). Conclusion. This is the first reported study of LRP as a prognostic marker in radiotherapy treated HNSCC suggesting it may play an important role not just in cancer drug resistance but also in radiation resistance. Of added interest is the difference in prognostic significance between the two subsites. References 1 Scheffer G.L., Wijngaard P.L., Flens M.J., et al. (1995) The drug resistance‐related protein LRP is the human major vault protein. Nature Med.1, 578–5822 Kozlov G., Vavelyuk O., Ovidiu M., et al. (2005) Solution structure of a two‐repeat fragment of major vault protein. J. Mol. Biol.7

Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.