A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

Proteomic analysis of Trypanosoma cruzi spliceosome complex.

The unicellular protists of the group Kinetoplastida include the genera Leishmania and Trypanosoma, which are pathogens of invertebrate and vertebrate animals. Despite their medical and economical importance, critical aspects of their biology such as specific molecular characteristics of gene expression regulation are just beginning to be deciphered. Gene expression regulation also depends on post-transcriptional processing steps, such as the trans-splicing process. Despite being widely used in trypanosomes, trans-splicing is a rare event in other eukaryotes. We sought to describe the protein composition of spliceosomes in epimastigotes of T. cruzi, the etiological agent of Chagas disease. We used two TAP-tagged proteins to affinity purify spliceosomes and analyzed their composition by mass spectrometry. Among the 115 identified proteins we detected conserved spliceosome components, as Sm and LSm proteins, RNA helicases, U2- and U5-snRNP specific proteins. Importantly, by comparing our data with proteomic data of human and T. brucei spliceosome complexes, we observed a core group of proteins common to all spliceosomes. By using amino acid sequence comparisons, we identified RNA-associated proteins that might be involved with splicing regulation in T. cruzi, namely the orthologous of WDR33, PABPCL1 and three different HNRNPs. Data are available via ProteomeXchange with identifier PXD018776.

Proteomic analysis of Trypanosoma cruzi spliceosome complex

The unicellular protists of the group Kinetoplastida include the genera Leishmania and Trypanosoma, which are pathogens of invertebrate and vertebrate animals. Despite their medical and economical importance, critical aspects of their biology such as specific molecular characteristics of gene expression regulation are just beginning to be deciphered. Gene expression regulation also depends on post-transcriptional processing steps, such as the trans-splicing process. Despite being widely used in trypanosomes, trans-splicing is a rare event in other eukaryotes. We sought to describe the protein composition of spliceosomes in epimastigotes of T. cruzi, the etiological agent of Chagas disease. We used two TAP-tagged proteins to affinity purify spliceosomes and analyzed their composition by mass spectrometry. Among the 115 identified proteins we detected conserved spliceosome components, as Sm and LSm proteins, RNA helicases, U2- and U5-snRNP specific proteins. Importantly, by comparing our data with proteomic data of human and T. brucei spliceosome complexes, we observed a core group of proteins common to all spliceosomes. By using amino acid sequence comparisons, we identified RNA-associated proteins that might be involved with splicing regulation in T. cruzi, namely the orthologous of WDR33, PABPCL1 and three different HNRNPs. Data are available via ProteomeXchange with identifier PXD018776.

Proteomic analysis of Trypanosoma cruzi spliceosome complex

The unicellular protists of the group Kinetoplastida include the genera Leishmania and Trypanosoma, which are pathogens of invertebrate and vertebrate animals. Despite their medical and economical importance, critical aspects of their biology such as specific molecular characteristics of gene expression regulation are just beginning to be deciphered. Gene expression regulation also depends on post-transcriptional processing steps, such as the trans-splicing process. Despite being widely used in trypanosomes, trans-splicing is a rare event in other eukaryotes. We sought to describe the protein composition of spliceosomes in epimastigotes of T. cruzi, the etiological agent of Chagas disease. We used two TAP-tagged proteins to affinity purify spliceosomes and analyzed their composition by mass spectrometry. Among the 115 identified proteins we detected conserved spliceosome components, as Sm and LSm proteins, RNA helicases, U2- and U5-snRNP specific proteins. Importantly, by comparing our data with proteomic data of human and T. brucei spliceosome complexes, we observed a core group of proteins common to all spliceosomes. By using amino acid sequence comparisons, we identified RNA-associated proteins that might be involved with splicing regulation in T. cruzi, namely the orthologous of WDR33, PABPCL1 and three different HNRNPs. Data are available via ProteomeXchange with identifier PXD018776.

Splicing and cancer: Challenges and opportunities.

Cancer arises from alterations in several metabolic processes affecting proliferation, growth, replication and death of cells. A fundamental challenge in the study of cancer biology is to uncover molecular mechanisms that lead to malignant cellular transformation. Recent genomic analyses revealed that many molecular alterations observed in cancers come from modifications in the splicing process, including mutations in pre-mRNA regulatory sequences, mutations in spliceosome components, and altered ratio of specific splicing regulators. While alterations in splice site preferences might generate alternative isoforms enabling different biological functions, these might also be responsible for nonfunctional isoforms that can eventually cause dysregulation in cellular processes. Molecular characteristics of regulatory sequences and proteins might also be important prognostic tools revealing a cancer-specific splicing pattern and linking splicing control to cancer development. The connection between cancer biology and splicing regulation is of primary importance to understand the mechanisms leading to disease and also to improve development of therapeutic approaches. Splicing modulation is being explored in new anti-cancer therapies and further investigation of targeted splicing factors is critical for the success of these strategies. This article is categorized under: RNA Processing > Splicing Mechanisms RNA-Based Catalysis > RNA Catalysis in Splicing and Translation RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

Human RNF113A participates of pre-mRNA splicing in vitro.

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Interaction paths promote module integration and network-level robustness of spliceosome to cascading effects.

The functionality of distinct types of protein networks depends on the patterns of protein-protein interactions. A problem to solve is understanding the fragility of protein networks to predict system malfunctioning due to mutations and other errors. Spectral graph theory provides tools to understand the structural and dynamical properties of a system based on the mathematical properties of matrices associated with the networks. We combined two of such tools to explore the fragility to cascading effects of the network describing protein interactions within a key macromolecular complex, the spliceosome. Using S. cerevisiae as a model system we show that the spliceosome network has more indirect paths connecting proteins than random networks. Such multiplicity of paths may promote routes to cascading effects to propagate across the network. However, the modular network structure concentrates paths within modules, thus constraining the propagation of such cascading effects, as indicated by analytical results from the spectral graph theory and by numerical simulations of a minimal mathematical model parameterized with the spliceosome network. We hypothesize that the concentration of paths within modules favors robustness of the spliceosome against failure, but may lead to a higher vulnerability of functional subunits, which may affect the temporal assembly of the spliceosome. Our results illustrate the utility of spectral graph theory for identifying fragile spots in biological systems and predicting their implications.

miR18a and miR19a Recruit Specific Proteins for Splicing in Thyroid Cancer Cells.

BACKGROUND: Thyroid cancer is one of the most frequent types of endocrine cancers. In most cases, thyroid cancers are caused by deregulated miRNA expression, especially involving the miR17-92 cluster. miR17-92 transcription is altered in several different tumor types including lymphoma, leukemia, and of the breast and thyroid. As an intronic cluster, miR17-92 must be processed during splicing and therefore interaction between microprocessor and spliceosome machineries is of major importance in understanding its expression. MATERIALS AND METHODS: We investigated the protein composition of spliceosomes assembled on pre-RNAs containing intronic miR18a and miR19a, components of the miR17-92 cluster, using mass spectrometry. RESULTS: Interestingly, we observed that proteins associated with intronic miR18a and miR19a are cell-specific, and are similar for both miRNAs analyzed. The only exception is the group of heterogeneous nuclear proteins that are commonly recruited by different cells. CONCLUSION: miRNA processing depends on cell-specific proteins and heterogeneous nuclear proteins have a general role in miRNA processing from introns.

Nestedness across biological scales.

Biological networks pervade nature. They describe systems throughout all levels of biological organization, from molecules regulating metabolism to species interactions that shape ecosystem dynamics. The network thinking revealed recurrent organizational patterns in complex biological systems, such as the formation of semi-independent groups of connected elements (modularity) and non-random distributions of interactions among elements. Other structural patterns, such as nestedness, have been primarily assessed in ecological networks formed by two non-overlapping sets of elements; information on its occurrence on other levels of organization is lacking. Nestedness occurs when interactions of less connected elements form proper subsets of the interactions of more connected elements. Only recently these properties began to be appreciated in one-mode networks (where all elements can interact) which describe a much wider variety of biological phenomena. Here, we compute nestedness in a diverse collection of one-mode networked systems from six different levels of biological organization depicting gene and protein interactions, complex phenotypes, animal societies, metapopulations, food webs and vertebrate metacommunities. Our findings suggest that nestedness emerge independently of interaction type or biological scale and reveal that disparate systems can share nested organization features characterized by inclusive subsets of interacting elements with decreasing connectedness. We primarily explore the implications of a nested structure for each of these studied systems, then theorize on how nested networks are assembled. We hypothesize that nestedness emerges across scales due to processes that, although system-dependent, may share a general compromise between two features: specificity (the number of interactions the elements of the system can have) and affinity (how these elements can be connected to each other). Our findings suggesting occurrence of nestedness throughout biological scales can stimulate the debate on how pervasive nestedness may be in nature, while the theoretical emergent principles can aid further research on commonalities of biological networks.

The network organization of protein interactions in the spliceosome is reproduced by the simple rules of food-web models.

The network structure of biological systems provides information on the underlying processes shaping their organization and dynamics. Here we examined the structure of the network depicting protein interactions within the spliceosome, the macromolecular complex responsible for splicing in eukaryotic cells. We show the interactions of less connected spliceosome proteins are nested subsets of the connections of the highly connected proteins. At the same time, the network has a modular structure with groups of proteins sharing similar interaction patterns. We then investigated the role of affinity and specificity in shaping the spliceosome network by adapting a probabilistic model originally designed to reproduce food webs. This food-web model was as successful in reproducing the structure of protein interactions as it is in reproducing interactions among species. The good performance of the model suggests affinity and specificity, partially determined by protein size and the timing of association to the complex, may be determining network structure. Moreover, because network models allow building ensembles of realistic networks while encompassing uncertainty they can be useful to examine the dynamics and vulnerability of intracelullar processes. Unraveling the mechanisms organizing the spliceosome interactions is important to characterize the role of individual proteins on splicing catalysis and regulation.

Cwc24p is a general Saccharomyces cerevisiae splicing factor required for the stable U2 snRNP binding to primary transcripts.

Splicing of primary transcripts is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamics of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to primary transcripts, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for stabilizing the U2 association with primary transcripts, and therefore, especially important for splicing of RNAs containing non-consensus branchpoint sequences.

The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

The NIP7 protein is required for accurate pre-rRNA processing in human cells.

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman-Bodian-Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S-80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.

Identification of archaeal proteins that affect the exosome function in vitro.

BACKGROUND: The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. RESULTS: Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. CONCLUSIONS: Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

Structural insights into the interaction of the Nip7 PUA domain with polyuridine RNA.

The conserved protein Nip7 is involved in ribosome biogenesis, being required for proper 27S pre-rRNA processing and 60S ribosome subunit assembly in Saccharomyces cerevisiae. Yeast Nip7p interacts with nucleolar proteins and with the exosome subunit Rrp43p, but its molecular function remains to be determined. Solution of the Pyrococcus abyssi Nip7 (PaNip7) crystal structure revealed a monomeric protein composed by two alpha-beta domains. The N-terminal domain is formed by a five-stranded antiparallel beta-sheet surrounded by three alpha-helices and a 310 helix while the C-terminal, a mixed beta-sheet domain composed by strands beta8 to beta12, one alpha-helix, and a 310 helix, corresponds to the conserved PUA domain (after Pseudo-Uridine synthases and Archaeosine-specific transglycosylases). By combining structural analyses and RNA interaction assays, we assessed the ability of both yeast and archaeal Nip7 orthologues to interact with RNA. Structural alignment of the PaNip7 PUA domain with the RNA-interacting surface of the ArcTGT (archaeosine tRNA-guanine transglycosylase) PUA domain indicated that in the archaeal PUA domain positively charged residues (R151, R152, K155, and K158) are involved in RNA interaction. However, equivalent positions are occupied by mostly hydrophobic residues (A/G160, I161, F164, and A167) in eukaryotic Nip7 orthologues. Both proteins can bind specifically to polyuridine, and RNA interaction requires specific residues of the PUA domain as determined by site-directed mutagenesis. This work provides experimental verification that the PUA domain mediates Nip7 interaction with RNA and reveals that the preference for interaction with polyuridine sequences is conserved in Archaea and eukaryotic Nip7 proteins.

Transcription analysis of pilS and xpsEL genes from Xylella fastidiosa.

Xylella fastidiosa is a xylem-limited phytopathogen responsible for diseases in several plants such as citrus and coffee. Analysis of the bacterial genome revealed some putative pathogenicity-related genes that could help to elucidate the molecular mechanisms of plant-pathogen interactions. In the present work, the transcription of three genes of the bacterium, grown in defined and rich media and also in media containing host plant extracts (sweet orange, 'ponkan' and coffee) was analyzed by RT-PCR. The pilS gene, which encodes a sensor histidine kinase responsible for the biosynthesis of fimbriae, was transcribed when the bacterium was grown in more complex media such as PW and in medium containing plant extracts. The xps genes (xpsL and xpsE) which are related to the type II secretion system were also detected when the bacterium was grown in rich media and media with 'ponkan' and coffee extracts. It was thus observed that pilS and xpsEL genes of X. fastidiosa can be modulated by environmental factors and their expression is dependent on the nutritional status of the growth medium.

Regulation of the htpX gene of Xylella fastidiosa and its expression in E. coli.

Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37 degrees C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37 degrees C or 45 degrees C in all culture media tested, but not at 28 degrees C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.