Cloning and characterization of the

The combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of protein complexes and large protein-interaction networks. Emerging AP–MS approaches promise a better understanding of protein-complex stoichiometry, structural organization and the dynamics of protein-complex composition. The versatile combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP–MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP–MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein–complex assembly.

http://bio.ijs.si/~krizaj/group/Struktura%20proteinov%202012/Seminarji-osnovna%20literatura/4.Gingras2007.pdf

Analysis of protein complexes using mass spectrometry

The composition of the soil microbial community can be altered dramatically due to association with individual plant species, and these effects on the microbial community can have important feedbacks on plant ecology. Negative plant-soil feedback plays primary roles in maintaining plant community diversity, whereas positive plant-soil feedback may cause community conversion. Host-specific differentiation of the microbial community results from the trade-offs associated with overcoming plant defense and the specific benefits associated with plant rewards. Accumulation of host-specific pathogens likely generates negative feedback on the plant, while changes in the density of microbial mutualists likely generate positive feedback. However, the competitive dynamics among microbes depends on the multidimensional costs of virulence and mutualism, the fine-scale spatial structure within plant roots, and active plant allocation and localized defense. Because of this, incorporating a full view of microbial dynamics is essential to explaining the dynamics of plant-soil feedbacks and therefore plant community ecology.

/pdf/microbial-population-and-community-dynamics-on-plant-roots-5azj3v37wr.pdf

Microbial population and community dynamics on plant roots and their feedbacks on plant communities.

http://www.bio.umass.edu/micro/klingbeil/590s/Reading/Stuart2005.pdf

Complex management: RNA editing in trypanosomes

https://www.cell.com/trends/parasitology/pdf/S1471-4922(03)00242-3.pdf

Chemotherapy of human African trypanosomiasis: current and future prospects

https://schnauferlab.bio.ed.ac.uk/PDFs/Schnaufer%20et%20al%20MolCell%202003.pdf

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3*-terminal uridylyl transferase, 3*-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an ;49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

/pdf/association-of-two-novel-proteins-tbmp52-and-tbmp48-with-the-2lw8tj95w8.pdf

Association of Two Novel Proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex

RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact. RNA editing in trypanosomes posttranscriptionally inserts and deletes uridylates (U’s) at multiple sites in most mitochondrial pre-mRNAs to produce mature mRNAs. U insertion and deletion are directed by guide RNAs (gRNAs) and are catalyzed by a macromolecular complex. Editing occurs by a series of enzymatic steps that include endoribonuclease, 3 terminal uridylyl transferase (TUTase), 3 exouridylylase, and RNA ligase activities (reviewed in references 3, 8, 21, and 23). Although editing can be extensive, with the insertion and deletion of numerous U’s, it is also very specific. The characteristics of the enzymatic activities contribute to this specificity (7), but noncatalytic proteins may be required for editing and may contribute to the specificity. RNA editing is catalyzed by a 20S ribonucleoprotein complex (2, 15), and identification of its components and the composition of the fully functional complex is at an early stage. Initial studies estimated that a complex that can catalyze at least some of the steps of editing in vitro contains 7 to 20 polypeptides (11, 14, 16). Two related proteins, TbMP52 and TbMP48, were identified by mass spectrometric analysis of purified editing complexes (14), and TbMP52 was shown to be essential for RNA editing and for survival of bloodstream forms in vivo and in vitro (19). In addition, TbMP52 and TbMP48 correspond to the larger and smaller adenylatable proteins in the RNA editing complex, respectively, and were found to be RNA ligases (12, 17, 19). TbMP52 corresponds to T. brucei V and T. brucei p52 and TbMP48 corresponds to T. brucei IV and T. brucei p48 (12, 17). The fully functional editing complex, which may consist of a catalytic core complex and accessory and regulatory factors may contain numerous proteins. Several other candidate proteins, some of which have RNA binding activities, have been described and may play a role in RNA editing (6, 9, 11, 13, 25). However, except for mHel61p (13), none of these proteins have been shown to play a direct role in editing. In this study, we describe the identification of four additional proteins that are present in the RNA editing complex by immunoprecipitation, mass spectrometric, and/or gradient sedimentation analyses. These four proteins have sequence similarities to each other and the three largest contain one or two C2H2 zinc finger motif(s). One protein was also shown to interact in vitro with the TbMP52 editing RNA ligase.

/pdf/four-related-proteins-of-the-trypanosoma-brucei-rna-editing-53unrs9x7o.pdf

Four Related Proteins of the Trypanosoma brucei RNA Editing Complex

RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5' fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5' fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.

Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing.

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.

/pdf/tbmp81-is-required-for-rna-editing-in-trypanosoma-brucei-43io9u2ccn.pdf

Setareh S. Palazzo

Papers

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.

Association of Two Novel Proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex

Four Related Proteins of the Trypanosoma brucei RNA Editing Complex

Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing.

TbMP81 is required for RNA editing in Trypanosoma brucei