Prediction of Gene Activity in Early B Cell Development Based on an Integrative Multi-Omics Analysis.

An increasingly common method for predicting gene activity is genome-wide chromatin immuno-precipitation of 'active' chromatin modifications followed by massively parallel sequencing (ChIP-seq). In order to understand better the relationship between developmentally regulated chromatin landscapes and regulation of early B cell development, we determined how differentially active promoter regions were able to predict relative RNA and protein levels at the pre-pro-B and pro-B stages. Herein, we describe a novel ChIP-seq quantification method (cRPKM) to identify active promoters and a multi-omics approach that compares promoter chromatin status with ongoing active transcription (GRO-seq), steady state mRNA (RNA-seq), inferred mRNA stability, and relative proteome abundance measurements (iTRAQ). We demonstrate that active chromatin modifications at promoters are good indicators of transcription and steady state mRNA levels. Moreover, we found that promoters with active chromatin modifications exclusively in one of these cell states frequently predicted the differential abundance of proteins. However, we found that many genes whose promoters have non-differential but active chromatin modifications also displayed changes in abundance of their cognate proteins. As expected, this large class of developmentally and differentially regulated proteins that was uncoupled from chromatin status used mostly post-transcriptional mechanisms. Strikingly, the most differentially abundant protein in our B-cell development system, 2410004B18Rik, was regulated by a post-transcriptional mechanism, which further analyses indicated was mediated by a micro-RNA. These data highlight how this integrated multi-omics data set can be a useful resource in uncovering regulatory mechanisms. This data can be accessed at: https://usegalaxy.org/u/thereddylab/p/prediction-of-gene-activity-based-on-an-integrative-multi-omics-analysis.

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion.

Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by approximately 20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 3'-U-exonuclease [3'-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 3'-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.

Trypanosoma brucei RNA editing: coupled cycles of U deletion reveal processive activity of the editing complex.

RNA editing in Trypanosoma brucei is posttranscriptional uridylate removal/addition, generally at vast numbers of pre-mRNA sites, but to date, only single editing cycles have been examined in vitro. We here demonstrate achieving sequential cycles of U deletion in vitro, with editing products confirmed by sequence analysis. Notably, the subsequent editing cycle is much more efficient and occurs far more rapidly than single editing cycles; plus, it has different recognition requirements. This indicates that the editing complex acts in a concerted manner and does not dissociate from the RNA substrate between these cycles. Furthermore, the multicycle substrate exhibits editing that is unexpected from a strictly 3'-to-5' progression, reminiscent of the unexpected editing that has been shown to occur frequently in T. brucei mRNAs edited in vivo. This unexpected editing is most likely due to alternate mRNA:guide RNA (gRNA) alignment forming a hyphenated anchor; its having only a 2-bp proximal duplex helps explain the prevalence of unexpected editing in vivo. Such unexpected editing was not previously reported in vitro, presumably because the common use of artificially tight mRNA:gRNA base pairing precludes alternate alignments. The multicycle editing and unexpected editing presented in this paper bring in vitro reactions closer to reproducing the in vivo editing process.

In Trypanosoma brucei RNA editing, TbMP18 (band VII) is critical for editosome integrity and for both insertional and deletional cleavages.

In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an approximately 20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the approximately 20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as approximately 10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the approximately 20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion cycle.

Trypanosome RNA editing is massive post-transcriptional U-insertion and U-deletion, which generates mature mRNA coding regions through cycles of endonuclease, terminal U transferase (TUTase) or 3'-U-exo, and ligase action. Both types of editing are thought to be catalyzed by distinct sets of proteins of a multiprotein complex, and no enzymatic activity of wild-type editing complex had been shown to function in both forms of editing. By examining the individual steps of the U-deletion cycle using purified editing complex, traditional mitochondrial extract, and rapidly prepared cell lysate, we here demonstrate that TbMP57 TUTase of U-insertion can act efficiently within a U-deletion cycle. When physiological UTP levels are provided, it adds U's to the upstream cleavage fragment after U-deletional endonuclease and 3'-U-exo action, but before rejoining by the U-deletional ligase, generating partial U-deletion products. TUTase activity in U-deletion was not previously appreciated since its detection requires UTP, which is not normally added to in vitro U-deletion reactions. Fractionation and RNAi analyses show this U-addition in U-deletion requires TbMP57 TUTase be present and competent for U-insertion; such U-addition does not occur with another mitochondrial TUTase that is separate from the basic editing complex. Efficient TbMP57 action in both U-insertion and U-deletion suggests these two editing forms may be less separate than generally envisioned. Should such promiscuous TUTase action also occur in vivo, it could explain why editing utilizes substantially fewer U-deletional than U-insertional events and why partial editing appears preferential in U-deletion.

In Trypanosoma brucei RNA editing, band II enables recognition specifically at each step of the U insertion cycle.

Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3'-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.