Finally, these data reveal a simple developmental trend in the oligodendrocyte lineage. Oligodendrocytes, which are primarily responsible for enwrapping neuronal axons with myelin sheaths, are a unique celltype in that their progenitor cells (OPCs) are widely distributed in the adult brain, where they actively proliferate and diffe- rentiate to generate new myelinating oligodendrocytes. Hence, we can detect gene expression and translation from different stages of oligodendrocyte development within homogenized brain tissue. Based on our analysis, OPC-specific genes are translated more efficiently than those of either newly formed or mature, myelinating oli- godendrocytes, which exhibit the lowest TE of the three. As shown in our statistical analysis in Additional file 4: Figure S4, the comparison between OPCs and myelinat- ing oligodendrocytes is very significant for highly trans- lated genes, as is the comparison between newly formed oligodendrocytes and myelinating oligodendrocytes. While one might expect myelinating oligodendrocytes to be less translationally active in comparison with OPCs because they are post-mitotic, their primary role in the brain is to produce large amounts of myelin, which is comprised mainly of proteins and lipids. Nonetheless, we found that most myelin genes have low TE compared with the overall median in the brain (log 2 (TE) = −0.02),
shikura, and Y. K. Shimizu, J. Virol. 70:3325–3329, 1996). These results suggest that virus with this 5 ⴕ NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap- independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had signif- icantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-freetranslation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.
Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specificcell types. This strategy relies on CRE recombinase–dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purifica- tion (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with celltype–spe- cific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific inju- ry signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.
Among the large number of upregulated genes de- tected within the positive cell population (Fig. 3c, d; Additional file 3: Figure S3a), many were known cni- docyte marker genes, including toxin (NEP-3, NEP-3-- like, NEP-4, and NEP-5) and structural protein-coding genes (NvNcol-1 and NvNcol-4). At the same time, there was downregulation of certain neuronal marker genes (FMRFamide and ELAV) that are known to lack expression in cnidocytes (Additional file 4: Figure S4; Additional file 6: Table S1). Though there was consistency with regard to the downregulation of FMRFamide and ELAV , we did not identify the upregulation of the afore- mentioned cnidocyte marker genes in the super-positive cell population (Additional file 5: Figure S5), which was enriched with mature cnidocytes. This can be explained by the fact that in mature cnidocytes, the capsule, which is a very tight polymer of various peptides and proteogly- cans, is already formed and the secretion of structural pro- teins is no longer required and is most probably wasteful. As a result, the expression of such genes diminishes with the maturation of cnidocytes. This is clearly evident when examining the expression of memOrange2 across various cell types. In our reporter line, memOrange2 was inte- grated into the genomic locus of NvNcol-3— the gene cod- ing a vital structural protein of the capsule (Fig. 1a). The expression of memOrange2 in the positive cell population was relatively higher than that in the negative cell popula- tion, but it dropped significantly in the super-positive population, where it was identified as a highly downregu- lated gene ( − 1.67 log 2 fold change; p value 0.0005)
Cap-independent translation of encephalomyocarditis virus (EMCV) RNA is controlled by a segment of the 5 * untranslated region termed the internal ribosomal entry site, or IRES. The IRES contains a series of stem-loop structural elements. The J and K stems (EMCV bases 682 to 795), near the center of the IRES, are well conserved among all cardio-, aphtho-, and hepatoviruses. We have examined the biological roles of these elements by constructing mutations within the J-K sequences of EMCV and testing the mutations for activity in translation, translation competition, UV cross-linking, and viral infectivity assays. Mutations near the helical junction of J and K proved severely detrimental to both cellular translation and cell-freetranslation of downstream cistrons. The same mutations reduced the ability of the IRES to compete for cellular factors in competition assays and reduced the infectivity of viral genomes carrying these lesions. A mutation in the terminal loop of J gave similar results. In contrast, mutations within the terminal loop of K had minimal impact on in vitro translation activity and IRES competitive ability. However, in vivo analysis of the K-loop mutations revealed deficiencies during cellular translation and further showed markedly reduced infectivity in HeLa cells. UV cross-linking experiments identified a 49-kDa protein which interacts strongly with the J-K region, but the identity of this protein and its contribution to IRES activity are unclear.
that are associated with vascular permeability. Interest- ingly, Cloutier et al. recently demonstrated that synovial vascular permeability observed in a murine experimental arthritis model was abrogated when platelets were de- pleted prior to the onset of arthritis . The authors note that this was an unexpected finding due to the clas- sical role of platelets in vessel maintenance, and may be the result of the severe inflammatory environment in the experimental arthritis model and the subsequent over- stimulation of platelets . Similarly, several reports note that antibody-mediated depletion of platelets can attenuate vascular permeability and leukocyte infiltration in an acute lung injury mouse model, via decreased endothelial cell adhesion molecule expression and me- diator release , and in a cecal ligation and puncture (CLP) murine model of sepsis, due to a decrease in both chemokine release and platelet-leukocyte interac- tions . Consistently, using both platelet depletion and CD40L-deficient animals, Rahman et al. identified platelet-derived CD40L as a driving force in the pathologic recruitment and infiltration of leukocytes into the lung in the CLP mouse model, thereby aiding in the development of lung injury and edema . Furthermore, Lapchak et al. also employed both platelet depletion and CD40- or CD40L-deficient mouse models of mesenteric ischemia/ reperfusion injury and observed lung damage only when platelets, CD40, or CD40L were present, whereas platelet- depleted mice and those deficient in CD40 or CD40L had a marked absence of vessel damage .
The DREAMM technique now adds to the growing number of research strategies in the neuroscience toolbox to assess in vivo brain function. Specifically, it provides, in contrast to other cur- rent techniques, the ability to measure in vivo time-dependent, regionally unbiased, whole-brain activity after cell-specific manip- ulations via a well-understood molecular process (glucose utili- zation). Importantly, while current imaging strategies have been limited to assessing brain function in immobilized animals, the unique kinetics of FDG allow time-dependent brain activity mea- sures that occur during the awake, freely moving state. DREAMM is most suited for studying behavioral profiles generated from ensembles of neurons than those relevant to the activity of a sin- gle cell. Moreover, DREAMM does not rely on complex surgical practices (chronic indwelling cannulas), which induce significant decreases in brain activity and inflammatory responses as well as cognitive deficits (25, 26). Since DREAMM is not limited to prob- ing cellular activity, it could also be used to provide insights into ed i.p. with vehicle or CNO and placed in an open-field arena.
Isolation and identification of HRV2 IRES sldV/VI-binding proteins. Since heterokaryon analyses implicated neuronal in- hibitors in HRV2 IRES incompetence, we searched for HRV2 IRES-binding proteins from neuroblastoma cells. We em- ployed IRES sldV/VI for our binding analysis, because genetic experiments mapped neuronal dysfunction to this portion of the IRES (9, 17, 18). To identify proteins present in neuroblas- toma RSW interacting with HRV2 IRES sldV/VI (Fig. 2A), we performed RNA affinity chromatography. RSW was loaded onto a Sepharose column coupled to sldV/VI. After extensive washes, bound proteins were eluted with a stepwise 300 to 1,000 mM KCl-buffer gradient, separated by SDS-PAGE, and silver stained (Fig. 2B). Six proteins with approximate masses of 130, 90, 65, 63, and 45 kDa were eluted from the column in the 400 mM to 600 mM KCl range at levels permitting recovery for identification by mass spectrophotometric analysis of tryp- tic peptide fragments (Fig. 2C). The deduced peptide se- quences (Table 1) and approximate molecular weights enabled unequivocal identification of p130 (RHA), p90 (DRBP76), p65 (IMP-1), p63 (hnRNP Q1), and p45 (NF45). RHA, DRBP76, IMP-1, and hnRNP Q1 contain RNA-binding motifs and have known roles in mRNA stability, localization, and translation.
In this study, we integrate high-quality cell-type-specific gene expression data and PPI data to build a collec- tion of 73 cell-type-specific interactomes and use these interactomes to create the first large-scale mapping of dis- eases to cell types. We use gene expression data from the FANTOM5 project , which represents the largest atlas of cell-type-specific gene expression produced to date. These data were created using primary cell samples rather than immortalized cell lines, resulting in higher- quality gene expression profiles . By comparing the clustering of sets of disease-associated genes across these cell-type-specific interactomes, we demonstrate that it is possible to use cell-type-specific interactomes to iden- tify the cell types in which a disease is most likely to be manifested. This approach is validated using text-mined disease–cell-type associations from the PubMed database. An implementation of the method described in this study and the 73 cell-type-specific interactomes are available to download [22, 23]. These resources will be useful in the identification of additional disease-associated cell types as more gene expression data become available, as well as in the development of tools better able to explore the etiology of a disease given its cellular con- text. Using this method, we identify known disease– cell-type associations and associations that warrant further study.
transfected with pSVC21 A4 may reflect the effect of the extra 11 amino acids (initiating from the upstream AUG) on pro- teolytic processing. The observation that A4 gag is present intracellularly in T cells but not in the supernatant is further evidence that the upstream novel initiation codon is being utilized, since translation from the upstream AUG would re- sult in a myristoylation-defective protein that would be ex- pected to be intracellularly confined. In light of the evidence from the T-cell transfections and the 21-day replication stud- ies, it is clear that the A4 provirus is able to generate gag protein intracellularly yet extracellular export is completely abolished. The A14 provirus is, however, unable to sustain protein synthesis in cells, as evidenced by the lack of gag p55, of intracellular cleavage products, or of extracellular reverse transcriptase activity. However, whether this is a direct result of the inability of the provirus to integrate into the host cell genome or due to the deletions of a packaging signal and splice donor site requires further elucidation. The difference in the abilities of COS and Jurkat cells to cleave pSVC21 A4 gag precursor is striking and emphasizes the cellular dependence of many viral processes and how results obtained with COS cells may not be relevant to events occurring in cells for which the virus is tropic.
We set out to determine whether the poly(A) binding protein, PABP, could be one of the factors additionally sequestered by the BDS for efficient trans-inhibition (Fig. 5). This hypothesis was based on the presence of a poly(A) mimic region embedded within the BDS, which can be functionally replaced by a poly(A) tail for translation in cis (3; Kneller, unpublished). We initially repressed translation of our BYDV-mimicking reporter construct (GfLUC) down to about 15% of its maximum efficiency, in presence of a 10-fold molar excess of sgRNA2 or 40-fold molar excess of BTE. We then measured restoration of translation by the increasing concentration of PABP added to the reaction. We hypothesized that if PABP is a limiting factor in the GfLUC repression and its addition should alleviate sgRNA2 inhibition, but not that mediated by BTE, which is not expected to sequester PABP due to the lack of the downstream sequences. Our results show that the addition of PABP was not able to reverse sgRNA2 trans-inhibition, or render GfLUC more efficient in translation. This suggests that the ability of sgRNA2 to repress translation more efficiently than BTE does not involve sequestration of PABP. The functionality of the PABP used in the assay was tested by the ability of PABP to restore translation of a capped, polyadenylated mRNA, which was repressed with the addition of a poly(A) tail to the reaction (data not shown).
To assess the conservation of these two CUG codons, and the potential for translation of the integrase C-ter- minal region as an independent protein in other gam- maretroviral lineages, we generated a codon-based alignment of pol genes (Additional file 1: Fig. S4). The CUG initiation codon of env-uORF-1 (Additional file 1: Fig. S4—highlighted in green) is conserved (or, in two cases, substituted with GUG or AUG) in most but not all lineages, and—where it is present—the context is always favourable for initiation: either with an A at − 3, or with a G at − 3 and a G at + 4. However, the lengths of the associated ORFs vary across lineages—ranging from 8 to 35 codons—and the corresponding amino acid sequences are also variable, suggesting that env-uORF-1 is unlikely to encode a functional peptide; nevertheless, its presence may serve to modulate env translation.
Here we used human neural stem cells (NSCs) derived from embryonic stem cells (ESCs) that have been bialleli- cally deleted for TSC2 by genome editing to study the cel- lular and molecular pathophysiology of TSC. This TSC in vitro model showed reduced neuronal maturation poten- tial and increased commitment to the astrocyte lineage, providing valuable insight for the study of TSC patient bi- opsies . Using RNA sequencing (RNA-Seq) and ribo- some profiling, we performed a comprehensive analysis of the genome-wide consequences of TSC2 loss on both transcription and translation. We detected a disease- relevant inflammatory response on the transcriptional level while translatome analysis demonstrated motif- dependent translational dysfunction of protein synthesis factors as well as increased production of angiogenic growth factors. Inhibition of mTOR signaling corrected the translation defects but not the inflammatory or angio- genic growth factor response, which were due to altered transcription. Thus we provide important insight into the molecular pathology of tuberous sclerosis and present an experimental system for future investigation of disease- modifying compounds beyond mTOR inhibitors and de- velopment of comprehensive therapies for TSC.
differential methylation analyses may harbor meaning- ful candidates for future studies. Indeed, our power study supports this idea (see the “Methods” section, Additional file 3: Figure S7). Consequently, we further analyzed sites that are ranked top 1000 in the differ- ential DNA methylation analysis between the brains of control vs. those from patients with schizophrenia (referred to as “top 1000” DMPs). We find that genes harboring the top 1000 szDMPs show enrichment for brain-related functions and diseases, as well as tran- scription factors, particularly those involved in chro- matin remodeling (Additional file 3: Figure S12). Given that the majority of the schizophrenia herita- bility is found below the significance thresholds of GWAS , we explored the association patterns at genome-wide SNPs. Top 1000 szDMPs tend to co- localize with genetic variants associated with schizo- phrenia but not with other mental or non-mental traits, mostly with genetic variants below the strict GWAS significance threshold but with moderate-to- high effect sizes (Fig. 4d). This result supports the role of brain DNA methylation in the genetic etiology of schizophrenia.