A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem af®nity puri®cation (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing-or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3¢ TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identi®ed by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids. Keywords: editosome/RNA editing/TAP/TUTase Introduction Uridine insertion/deletion RNA editing is a post-transcriptional RNA modi®cation phenomenon that occurs in the mitochondrion of kinetoplastid protists . The mechanism involves the initial hybridization to an mRNA of a complementary guide RNA (gRNA) which guides a speci®c endonuclease cleavage at the ®rst editing site . This is followed by either deletion of the unpaired uridines from the cleavage fragment or the 3¢ addition to the mRNA 5¢ cleavage fragment, hybridization of the added Us to the guiding nucleotides in the gRNA, and religation of the two mRNA cleavage fragments. Each gRNA speci®es the 3¢ to 5¢ editing of a small number of sites and, in the case of a multiple gRNA-mediated editing domain, creates the anchor sequence for hybridization of the adjacent upstream gRNA, thus producing an overall 3¢ to 5¢ progression of editing. A minimal non-progressive editing activity at one or two sites has been demonstrated in vitro using crude or partially puri®ed mitochondrial extract, and the reaction was shown to involve high molecular weight RNP complexes (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Kable et al., 1996;Seiwert et al., 1996). The mechanism described above was proposed >12 years ago , and was veri®ed experimentally in 1996 for both Trypanosoma brucei and Leishmania tarentolae (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Seiwert et al., 1996). However, progress in the identi®cation of speci®c proteins involved in editing has been hampered by their low abundance and by the low ef®ciency of the in vitro editing assays. A seven polypeptide complex from T.brucei mitochondria that supported in vitro insertion and deletion editing was isolated by two chromatographic steps and was proposed to represent a core editing complex (Rusche et al., 1997). An~20 polypeptide complex with similar activities was isolated in another laboratory by a similar fractionation (Panigrahi et al., 2001a,b).The genes for several of the major components of these complexes have been identi®ed, but only a few proteins so far have been ascribed ...

Engineered nanomaterials (ENMs) including multiwall carbon nanotubes (MWCNTs) and rare earth oxide (REO) nanoparticles, which are capable of activating the NLRP3 inflammasome and inducing IL-1β production, have the potential to cause chronic lung toxicity. Although it is known that lysosome damage is an upstream trigger in initiating this pro-inflammatory response, the same organelle is also an important homeostatic regulator of activated NLRP3 inflammasome complexes, which are engulfed by autophagosomes and then destroyed in lysosomes after fusion. Although a number of ENMs have been shown to induce autophagy, no definitive research has been done on the homeostatic regulation of the NLRP3 inflammasome during autophagic flux. We used a myeloid cell line (THP-1) and bone marrow derived macrophages (BMDM) to compare the role of autophagy in regulating inflammasome activation and IL-1β production by MWCNTs and REO nanoparticles. THP-1 cells express a constitutively active autophagy pathway and are also known to mimic NLRP3 activation in pulmonary macrophages. We demonstrate that, while activated NLRP3 complexes could be effectively removed by autophagosome fusion in cells exposed to MWCNTs, REO nanoparticles interfered in autophagosome fusion with lysosomes. This leads to the accumulation of the REO-activated inflammasomes, resulting in robust and sustained IL-1β production. The mechanism of REO nanoparticle interference in autophagic flux was clarified by showing that they disrupt lysosomal phosphoprotein function and interfere in the acidification that is necessary for lysosome fusion with autophagosomes. Binding of LaPO4 to the REO nanoparticle surfaces leads to urchin-shaped nanoparticles collecting in the lysosomes. All considered, these data demonstrate that in contradistinction to autophagy induction by some ENMs, specific materials such as REOs interfere in autophagic flux, thereby disrupting homeostatic regulation of activated NLRP3 complexes.

A number of mitochondrial proteins have been identified in Leishmania sp. and Trypanosoma brucei that may be involved in U-insertion/deletion RNA editing. Only a few of these have yet been characterized sufficiently to be able to assign functional names for the proteins in both species, and most have been denoted by a variety of species-specific and laboratory-specific operational names, leading to a terminology confusion both within and outside of this field. In this review, we summarize the present status of our knowledge of the orthologous and unique putative editing proteins in both species and the functional motifs identified by sequence analysis and by experimentation. An online Supplemental sequence database (http://164.67.60.200/ proteins/protsmini1.asp) is also provided as a research resource.

Higher expression of human telomerase reverse transcriptase (hTERT) and subsequent activation of telomerase occur during cellular immortalization and are maintained in cancer cells. To understand the mode of hTERT expression in cancer cells, we identified cancer-specific trans-regulatory proteins that interact with the hTERT promoter, using the promoter magnetic precipitation assay coupled to mass spectrometry (PMS-MS). The identified proteins include MutS homologue 2 (MSH2), heterogeneous nuclear ribonucleoprotein (hnRNP) D, hnRNP K, and Grainyhead-like 2 (GRHL2). We noticed higher expression of these proteins in human oral squamous cell carcinoma (OSCC) cells than in normal cells, which do not exhibit telomerase activity. Knockdown of MSH2, hnRNP D and GRHL2 resulted in notable reduction of the hTERT promoter activity in tested cancer cells. Silencing of the above genes resulted in the significant reduction of telomerase activity in OSCC cells. Interestingly, among the four identified genes, silencing of GRHL2 was essential in reducing telomerase activity and viability of tested cancer cells. These results suggest a possible role of GRHL2 in telomerase activation during cellular immortalization.

Uridine insertion͞deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3 end of the 5 mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3-5 exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3 overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3 overhanging Us from the bridged mRNA 5 cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.ligase ͉ trypanosomes ͉ REX1 ͉ editing U ridine insertion͞deletion RNA editing is a posttranscriptional RNA modification phenomenon that occurs in the mitochondria of kinetoplastid protists (1). The insertion and deletion of Us into transcripts of 12 mitochondrial-encoded cryptogenes is mediated by guide RNAs (gRNAs) that hybridize downstream of the editing sites and recruit several protein complexes that interact via RNA. The RNA ligase-containing complex (L-complex) from both Leishmania sp. and Trypanosoma brucei contains Ϸ16 proteins, which have been labeled LC-X (for L-complex protein) or MP-X (for mitochondrial protein), respectively (2, 3), by relative gel mobility. These proteins include the LC-2 (MP100) and LC-3 (MP99) ''Exoendophos''-Pfam motif proteins (4), the RNA editing ligase 1 (REL1) (5-7) and RNA editing ligase 2 (REL2), and the RNA editing 3Ј terminal uridylyltransferase (TUTase) 2 (RET2) proteins, among others. Editing involves an initial cleavage of the mRNA transcript just upstream of the mRNA-gRNA anchor duplex, which is followed by either an addition of Us to the 3Ј end of the 5Ј fragment or a deletion of non-base-paired Us from the 3Ј end of the 5Ј fragment. The two fragments are then ligated, thereby extending the mRNA-gRNA duplex. The process repeats at the next upstream editing site, and, after completion of the editing mediated by a single gRNA, in some cases, additional gRNAs hybridize to the edited sequences and mediate overlapping blocks of editing, extending the edited region further upstream (8). The RET2 TUTase in the L-complex was shown to be responsible for the gRNA-mediated addition of Us to the editing sites, and the RET1 TUTase, which is not a component of the L-complex but interacts via RNA, is responsible for the addition of Us to the 3Ј end of the gRNAs (9, 10).Heterologous expression of properly folded active editing enzymes has proven difficult. The only recombinant...

Oxidative stress plays an important role in the development of airway inflammation and hyperreactivity in asthma. The identification of oxidative stress markers in bronchoalveolar lavage fluid (BALF) and lung tissue from ovalbumin (OVA) sensitized mice could provide new insight into disease pathogenesis and possible use of antioxidants to alleviate disease severity. We used twodimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the impact of the thiol antioxidant, N-acetylcysteine (NAC), on protein expression in a murine OVA model. At least six proteins or protein families were found to be significantly increased in BALF from OVA-challenged mice compared to a control group: chitinase 3-like protein 3 (Ym1), chitinase 3-like protein 4 (Ym2), acidic mammalian chitinase (AMCase), pulmonary surfactant-associated protein D (SP-D), resistin-like molecule α (RELMα) or "found in inflammatory 1" (FIZZ1), and haptoglobin α-subunit. A total of 9 proteins were significantly increased in lung tissue from the murine asthma model, including Ym1, Ym2, FIZZ1, and other lung remodeling-related proteins. Western blotting confirmed increased Ym1/Ym2, SP-D, and FIZZ1 expression measured from BAL fluid and lung tissue from OVA-challenged mice. Intraperitoneal NAC administration prior to the final OVA challenge inhibited Ym1/Ym2, SP-D, and FIZZ1 expression in BALF and lung tissue. The oxidative stress proteins, Ym1/Ym2, FIZZ1 and SP-D, could play an important role in the pathogenesis of asthma and may be useful oxidative stress markers.

Background Cardiac maturation during perinatal transition of heart is critical for functional adaptation to hemodynamic load and nutrient environment. Perturbation in this process has major implications in congenital heart defects (CHDs). Transcriptome programming during perinatal stages is important information but incomplete in current literature, particularly, the expression profiles of the long noncoding RNAs (lncRNAs) are not fully elucidated. Methods and Results From comprehensive analysis of transcriptomes derived from neonatal mouse heart left and right ventricles, a total of 45,167 unique transcripts were identified, including 21,916 known and 2,033 novel lncRNAs. Among these lncRNAs, 196 exhibited significant dynamic regulation along maturation process. By implementing parallel weighted gene co-expression network analysis (WGCNA) of mRNA and lncRNA datasets, several lncRNA modules coordinately expressed in a developmental manner similar to protein coding genes, while few lncRNAs revealed chamber specific patterns. Out of 2,262 lncRNAs located within 50 KBs of protein coding genes, 5% significantly correlate with the expression of their neighboring genes. The impact of Ppp1r1b-lncRNA on the corresponding partner gene Tcap was validated in cultured myoblasts. This concordant regulation was also conserved in human infantile hearts. Furthermore, the Ppp1r1b-lncRNA/Tcap expression ratio was identified as a molecular signature that differentiated CHD phenotypes. Conclusions The study provides the first high-resolution landscape on neonatal cardiac lncRNAs and reveals their potential interaction with mRNA transcriptome during cardiac maturation. Ppp1r1b-lncRNA was identified as a regulator of Tcap expression with dynamic interaction in postnatal cardiac development and CHDs.

Uridine (U)-insertion͞deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model Udeletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNAediting nuclease 1.mitochondria involves the participation of at least three RNAlinked multiprotein complexes, the Ϸ19-polypeptide ligasecontaining core editing complex (L-complex), the mitochondrial RNA-binding protein (MRP) complex, and the RNA editing 3Ј terminal uridylyl transferase (TUTase) (RET)1 complex(es) (1-3). L-complex proteins that have been expressed in active form and characterized include the RNA editing ligases 1 and 2 (REL1 and REL2) (4-7), the RNA editing 3Ј-5Ј U-specific exonucleases (REX1 and REX2) (8-10), and the RET2Ј3Ј TUTase (11-13). Characterization of the protein components of the L-complex led to several nuclease candidates: LC6A, or MP61, MP90, and MP67 contain RNase III motifs, and LC8, or MP44, has a highly diverged RNase III motif (11,(14)(15)(16). Although TbMP90 and TbMP61 were stated in recent reviews (1, 16) to also contain double-strand RNA-binding motifs, such motifs are not readily identifiable (L.S., unpublished data). In addition, the MP42 L-complex protein, which has only zinc finger (ZnF C2H2) and single-strand RNA-binding (SSB) motifs, nevertheless exhibited both exonuclease and endonuclease activities, but the activities identified did not show the required specificity, and the role of this protein remains an open question (17). Trotter et al. (18) and Carnes et al. (19) recently provided in vivo evidence for a specific role in cleavage at mRNA U-deletion and U-insertion editing sites for, respectively, the essential TbMP90 and TbMP61 RNase III motif-containing proteins. It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18,19).In this study, we provide both indirect and direct evidence for a role of the MP90 L-complex protein in the initial cleavage at preedited mRNA U-deletion editing sites, and we sh...