Viral Infection and Stress Affect Protein Levels of Dicer 2 and Argonaute 2 in Drosophila melanogaster.

The small interfering RNA (siRNA) pathway of Drosophila melanogaster, mainly characterized by the activity of the enzymes Dicer 2 (Dcr-2) and Argonaute 2 (Ago-2), has been described as the major antiviral immune response. Several lines of evidence demonstrated its pivotal role in conferring resistance against viral infections at cellular and systemic level. However, only few studies have addressed the regulation and induction of this system upon infection and knowledge on stability and turnover of the siRNA pathway core components transcripts and proteins remains scarce. In the current work, we explore whether the siRNA pathway is regulated following viral infection in D. melanogaster. After infecting different fly strains with two different viruses and modes of infection, we observed changes in Dcr-2 and Ago-2 protein concentrations that were not related with changes in gene expression. This response was observed either upon viral infection or upon stress-related experimental procedure, indicating a bivalent function of the siRNA system operating as a general gene regulation rather than a specific antiviral system.

DHX37 Impacts Prognosis of Hepatocellular Carcinoma and Lung Adenocarcinoma through Immune Infiltration.

BACKGROUND: RNA helicases have various essential functions in basically all aspects of RNA metabolism, not only unwinding RNA but also disturbing the interaction of RNA with proteins. Recently, RNA helicases have been considered potential targets in cancers. So far, there has been no detailed investigation of the biological functions of RNA helicase DHX37 in cancers. OBJECTIVE: We aim to identify the prognostic value of DHX37 associated with tumor microenvironments in cancers. METHODS: DHX37 expression was examined via the Oncomine database and Tumor Immune Estimation Resource (TIMER). We explored the prognostic role of DHX37 in cancers across various databases. Coexpression genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and fundamental regulators were performed via LinkedOmics. Confirming the prognostic value of DHX37 in liver hepatocellular carcinoma (LIHC) and lung adenocarcinoma (LUAD), we explored the role of DHX37 in infiltrated lymphocytes in cancers using the Gene Expression Profiling Interactive Analysis (GEPIA) and TIMER databases. RESULTS: Through GO and KEGG analyses, expression of DHX37 was also correlated with complex function-specific networks involving the ribosome and RNA metabolic signaling pathways. In LIHC and LUAD, DHX37 expression showed significant positive correlations with markers of Tregs, myeloid-derived suppressor cells (MDSCs), and T cell exhaustion, contributing to immune tolerance. CONCLUSION: These results indicate that DHX37 can serve as a prognostic biomarker in LIHC and LUAD while having an important role in immune tolerance by activating the function of Tregs, MDSC, and T cell exhaustion.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

[Dectection of G3BP and CD44v6 in the tissues of laryngeal squamous cell carcinoma and their clinical significance].

Objective To study the expressions of RNA-binding Ras-GAP SH3 binding protein (G3BP) and tumor stem cell marker CD44v6 in laryngeal squamous cell carcinoma and their correlations with angiogenesis. Methods We collected the cancer tissues and corresponding paracancerous tissues from 56 patients with laryngeal squamous cell carcinoma. The expressions of G3BP and CD44v6 proteins were detected by Western blotting in cancer tissues and corresponding paracancerous tissues; the expressions of G3BP, CD44v6 and vascular endothelial growth factor A (VEGF-A) were tested by immunohistochemistry. Thereafter, we compared the positive expression rates of G3BP and CD44v6 between in cancer tissues and in normal tissues, analyzed the correlations between the expressions of G3BP, CD44v6 and the laryngeal squamous cell carcinoma features as well as their correlations with microvessel density (MVD) that was determined by FVIIIAg immunohistochemistry. Results Western blotting showed that the expressions of G3BP and CD44v6 proteins in the laryngeal squamous cell carcinoma were higher than those in the paracancerous tissues. Immunohistochemistry showed that compared with the paracancerous tissues, G3BP, CD44v6 and VEGF-A expressions (the positive rates are 58.9%, 53.6%, 46.4%, respectively) were higher in cancer tissues. The positive rates of G3BP and CD44v6 in cancer tissues were related with the clinical stage, recurrence or metastasis, and lymph node metastasis of laryngeal squamous cell carcinoma, but had nothing to do with patients' age and tumor size. Pearson correlation analysis showed the expressions of both G3BP and CD44v6 were positively correlated with VEGF-A (r=0.741, r=0.756). MVD values were significantly higher in the G3BP and CD44v6 positive cases than in paracancerous tissues, but there was no difference in MVD between those without G3BP and CD44v6 positive expressions and the paracancerous tissues. Conclusion The positive expression rates of G3BP and CD44v6 in laryngeal squamous cell carcinoma tissues are very high, and they have a close relationship with the clinical prognosis. They may raise the VEGF-A expression so as to promote angiogenesis, and then accelerate the development of the laryngeal squamous cell carcinoma.

Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

Impact of three inactivated bovine viral diarrhoea virus vaccines on bulk milk p80 (NS3) ELISA test results in dairy herds.

Bovine viral diarrhoea virus (BVDV) is endemic in many countries and vaccines are used as a component of control and eradication strategies. Surveillance programmes to detect exposure to BVDV often incorporate the use of bulk milk (BM) testing for antibodies against BVDV p80 (NS3), but vaccination can interfere with these results. The aim of this study was to evaluate whether BVDV vaccines would confound BM testing for specific antibodies in a nationally representative group of commercial dairy farms in the Republic of Ireland. A total of 256 commercial dairy herds were included in the statistical analysis. Quarterly BM or serum samples from selected weanling heifers (unvaccinated homeborn youngstock) were assessed by ELISA for antibodies against the BVDV p80 subunit and whole virus. Wilcoxon rank-sum and receiver operating characteristic (ROC) analyses were used to examine differences among groups vaccinated with one of three commercially available inactivated BVDV vaccines. Two of the three vaccines showed evidence of interference with ELISA testing of BM samples. ROC analysis highlighted that one vaccine did not reduce the discriminatory power of the BVDV p80 ELISA for identification of herds with evidence of recent BVDV circulation, when compared with unvaccinated herds; thus, administration of this vaccine would allow uncomplicated interpretation of BM ELISA test results in vaccinated seropositive herds. Seasonal differences in BM antibody results were identified, suggesting that the latter half of lactation is the most suitable time for sampling dairy herds containing predominantly spring calving cows. The results of the present study are likely to prove useful in countries allowing vaccination during or following BVDV eradication, where BM testing is required as part of the surveillance strategy.

AGO3 Slicer activity regulates mitochondria-nuage localization of Armitage and piRNA amplification.

In Drosophila melanogaster the reciprocal "Ping-Pong" cycle of PIWI-interacting RNA (piRNA)-directed RNA cleavage catalyzed by the endonuclease (or "Slicer") activities of the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) has been proposed to expand the secondary piRNA population. However, the role of AGO3/Aub Slicer activity in piRNA amplification remains to be explored. We show that AGO3 Slicer activity is essential for piRNA amplification and that AGO3 inhibits the homotypic Aub:Aub Ping-Pong process in a Slicer-independent manner. We also find that expression of an AGO3 Slicer mutant causes ectopic accumulation of Armitage, a key component in the primary piRNA pathway, in the Drosophila melanogaster germline granules known as nuage. AGO3 also coexists and interacts with Armitage in the mitochondrial fraction. Furthermore, AGO3 acts in conjunction with the mitochondria-associated protein Zucchini to control the dynamic subcellular localization of Armitage between mitochondria and nuage in a Slicer-dependent fashion. Collectively, our findings uncover a new mechanism that couples mitochondria with nuage to regulate secondary piRNA amplification.

Quantitative proteomic analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 (GBF1) in dengue infection.

Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.

Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.

Rational design of modular allosteric aptamer sensor for label-free protein detection.

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein-aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein-aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.

Transient elimination of circulating bovine viral diarrhoea virus by colostral antibodies in persistently infected calves: a pitfall for BVDV-eradication programs?

Infections with bovine viral diarrhoea virus (BVDV) cause substantial economic losses to cattle industries. Rapid detection of persistently BVDV infected (PI) calves is of utmost importance for the efficacy of BVDV control programs. Blood and ear skin biopsy samples are conveniently used for early mass screening of newborns. However, little is known about the impact of colostral antibodies on the outcome of relevant analyses. Here, we rigorously tested a series of samples obtained from five colostrum-fed PI calves from birth until they reached the status of seronegativity for NS3-specific antibodies. We comparatively quantified virus loads in blood samples and dried skin biopsies as detected with BVDV-NS3-, -Erns-capture ELISA and RT-qPCR. Monitoring of NS3-positive leukocytes was done with flow cytometry. Within seven days after colostrum intake, BVDV infected leukocytes disappeared for a three- to eight-week period. Immediately after colostrum ingestion, detectable Erns antigen levels dropped 10-100-fold in biopsy samples and in sera detection of Erns failed for one to two weeks. Virus demonstration in biopsy samples with a NS3-antigen-ELISA failed until days 90-158 after birth. Specific antibodies against BVDV also impaired the detection of viral RNA in leukocytes and blood. Mean RNA levels of the five calves were reduced in sera 2.500-fold and in leukocytes 400-fold, the lowest values were at week three of live. In contrast, levels of measurable viral RNA in biopsy samples remained constant during the observation period.

Phosphatase activity in barley proteins tightly bound to DNA and its development-dependent changes.

The tightly bound proteins (TBPs), a protein group that remains attached to DNA either covalently or noncovalently after deproteinization, have been found in numerous eukaryotic species. Some TBPs isolated from mammalian and yeast cells possess phosphatase or kinase activity. The aim of this study was to characterize further TBPs in barley (Hordeum vulgare) cells. The spectra of TBPs varied in different organs of barley shoots (first leaves, coleoptile, and roots) and at different developmental stages of the plant. Some barley TBPs manifested phosphatase, probably Ser/Thr or dual Ser/Thr/Tyr activity. MALDI-TOF mass spectrometry of barley TBPs identified several proteins involved in chromatin rearrangement and regulation processes, including transcription factors, serpins, protein phosphatases and protein kinases, RNA helicases, and DNA topoisomerase II.

Nuage proteins: their localization in subcellular structures of spermatogenic cells as revealed by immunoelectron microscopy.

Chromatoid body (CB) was identified as granules stained by basic dye 130 years ago and called by various names. Electron microscopy revealed that the CB belonged to nuage (cloud in French) specific for germ cells. We described the localization of several proteins, including RNA helicases, in the nuage compartments classified into six types and in several spermatogenic cell-specific structures. All the proteins examined were detected in the nuage, including the CB with different staining intensities. Several proteins were localized to non-nuage structures, suggesting that these nuage proteins structures are related to nuage function.

The complete genome sequence of a single-stranded RNA virus from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois).

The complete genome sequence of a single-stranded RNA virus infecting the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by sequencing cDNA prepared from insects collected from the Mississippi Delta. The 9655 nucleotide positive-sense single-stranded RNA genome of the L. lineolaris single-stranded RNA virus (LyLV-1) contained a single open reading frame of 8958 nucleotides encoding a 2986 amino acid genome polypeptide. The open reading frame was flanked by untranslated regions of 603 and 69 nucleotides at the 5'- and 3'- ends of the genome, respectively. Database searches and homology based modeling was used to identify four capsid proteins (VP1-VP4), helicase/AAA-ATPase, cysteine protease (C3P), protease 2A, and the RNA-directed RNA polymerase (RdRp). In addition, a region with weak similarity to the eukaryotic structural maintenance of chromosome (SMC) domain was identified near the amino-terminal of the polyprotein and adjacent to the VP1 domain. The amino acid sequence of LyLV-1 was approximately 44.4% similar to that of sacbrood virus (SBV) of the honey bee. The genomic organization of both viruses showed remarkable similarity with the exception of highly divergent amino acid regions flanking fairly conserved structural and non-structural polypeptide regions. High similarity to the SBV genome and similarities in the genome organization and amino acid sequence with the viruses of the family Iflaviridae suggested that LyLV-1 was a novel member of this family. Virus particles were 39 nm in diameter and appeared to transmit vertically via eggs. Although this virus may only cause covert infections under normal conditions, the potential for using this virus in biological control of L. lineolaris is discussed.

Interaction profiling identifies the human nuclear exosome targeting complex.

The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.

Multilayer fluorescence optically encoded beads for protein detection.

An easy preparation method of multilayer fluorescence optically encoded beads for protein detection is presented. The beads, which consist of multicolored layers, are made from amino polyethylene glycol grafted polystyrene (PS-g-PEG) beads by using several fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine via controlling diffusion of an Fmoc-protecting group after HCl solution swelling. A biotin, glutathione S-transferase (GST) antibody, and an RNA aptamer that specifically recognize streptavidin, GST antigen, and hepatitis C virus (HCV) helicase are introduced to the optically encoded beads and monitored for their binding activity to the target molecules. After binding, the ligands are identified easily by their color codes.

Antiviral activity of geneticin against bovine viral diarrhoea virus.

BACKGROUND: Aminoglycoside G418 is commonly used to generate stable replicons for RNA viruses, such as hepatitis C virus, West Nile virus, and bovine viral diarrhoea virus (BVDV). This precludes testing 6418's own antiviral activities against those viruses. Here, we report antiviral activity of 6418 against BVDV. METHODS: Cell viability and virus yield reduction assays were used to investigate antiviral effects of G418 against BVDV. The expression of viral proteins and RNA were determined by western blot and real-time quantitive PCR, respectively. RESULTS: We demonstrated that G418 (50% cytotoxicity concentration of 400 microg/ml) improved cell viability of Madin-Darby bovine kidney cells infected with a cytopathic strain of BVDV (NADL) in a dose-dependent manner with 50% effective concentration of 4 microg/ml. Interestingly, close structural analogues with known properties as translation inhibitors similar to G418 - kanamycin and gentamicin - had no antiviral activity against BVDV. In addition, 6418 inhibits virus yield of two different strains of BVDV (NADL and NY-1) without affecting viral RNA replication and translation or viral NS3 protein processing. CONCLUSION: Our data indicate that antiviral activity of G418 could result from interference with either the assembly or release of active virus, rather than the regulation of viral translation and replication. Thus, we propose the use of chemical analogues of G418 as antiviral therapeutics for treatment of viral diseases associated with the Flaviviridae family, such as hepatitis C virus, dengue virus, yellow fever virus, West Nile virus and others.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.

Localization of Vasa mRNA during early cleavage of the snail Ilyanassa.

Members of the Vasa family of helicases are specifically localized to germ line lineages in embryos of many animal groups and, in some cases, have been shown to be required for germ line formation. Despite considerable attention to the embryology of gastropod molluscs, the germ line has not been identified in the early cleavage stages of these embryos. We have cloned a Vasa ortholog in the snail Ilyanassa and examined the distribution of IoVasa mRNA during early cleavage. Initially, the transcript is present in all cells and non-specifically localized to centrosomes in a subset of cells. The IoVasa mRNA becomes progressively more enriched in the dorsal quadrant of the embryo, and then becomes restricted to particular cells in the 4d lineage. At the 64-cell stage, IoVasa mRNA is detected in 4dL11, 4dL12, 4dR11, and 4dR12. Following another round of division in the 4d lineage, the mRNA is restricted to two cells: 4dL121 and 4dR121. By the 108-cell stage, IoVasa mRNA is no longer detectable. Because the germ line is thought to arise from the 4d lineage in spiralians, these data are consistent with the hypothesis that the Ilyanassa germ line is marked by inheritance of IoVasa and derived from the cells 4dL121 and 4dR121. Alternatively, IoVasa may be required in somatic lineages where it is expressed, and the germ line may be specified later in development.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.