Differential Effects of Arginine Methylation on RBP16 mRNA Binding, Guide RNA (gRNA) Binding, and gRNA-containing Ribonucleoprotein Complex (gRNP) Formation

Goulah, Christopher C.; Read, Laurie K.

doi:10.1074/jbc.m609485200

Cited by 31 publications

(43 citation statements)

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“…RNA editing initiates with formation of the gRNA/pre-mRNA hybrid, prior to pre-mRNA cleavage. Evidence from in vitro RNA editing assays indicates that RBP16 stimulates editing at, or prior to, the cleavage of pre-mRNA (Miller et al 2006), and RBP16 interacts with both gRNA and mRNA in vivo (Hayman and Read 1999;Goulah and Read 2007). In this paper, we have demonstrated that RBP16 has RNA annealing and unwinding activity.…”

Section: Discussionmentioning

confidence: 61%

“…In addition to its in vivo association with gRNAs, it has been shown to associate with 9S and 12S rRNAs and a subset of mRNAs in T. brucei mitochondria (Hayman and Read 1999;Militello et al 2000;Goulah and Read 2007). RBP16 was the first characterized RNA editing accessory factor, and both in vivo and in vitro assays demonstrate a role for RBP16 in stimulation of RNA editing (Pelletier and Read 2003;Miller et al 2006).…”

Section: Discussionmentioning

confidence: 99%

“…In vivo, through both immunoprecipitation and in organello cross-linking studies, RBP16 was shown to bind z30% of the total gRNAs (Hayman and Read 1999;Militello et al 2000). Additionally, RBP16 associates with 9S and 12S rRNAs and certain mitochondrial mRNAs (Hayman and Read 1999;Goulah and Read 2007).…”

Section: Introductionmentioning

confidence: 99%

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gRNA/pre-mRNA annealing and RNA chaperone activities of RBP16

Ammerman¹,

Fisk²,

Read³

2008

RNA

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Editing in trypanosomes involves the addition or deletion of uridines at specific sites to produce translatable mitochondrial mRNAs. RBP16 is an accessory factor from Trypanosoma brucei that affects mitochondrial RNA editing in vivo and also stimulates editing in vitro. We report here experiments aimed at elucidating the biochemical activities of RBP16 involved in modulating RNA editing. In vitro RNA annealing assays demonstrate that RBP16 significantly stimulates the annealing of gRNAs to cognate pre-mRNAs. In addition, RBP16 also facilitates hybridization of partially complementary RNAs unrelated to the editing process. The RNA annealing activity of RBP16 is independent of its high-affinity binding to gRNA oligo(U) tails, consistent with the previously reported in vitro editing stimulatory properties of the protein. In vivo studies expressing recombinant RBP16 in mutant Escherichia coli strains demonstrate that RBP16 is an RNA chaperone and that in addition to RNA annealing activity, it contains RNA unwinding activity. Our data suggest that the mechanism by which RBP16 facilitates RNA editing involves its capacity to modulate RNA secondary structure and promote gRNA/pre-mRNA annealing.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

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gRNA/pre-mRNA annealing and RNA chaperone activities of RBP16

Ammerman¹,

Fisk²,

Read³

2008

RNA

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“…Moreover, arginine methylation was shown to decrease the capacity of RBP16 to associate with gRNA. This is not a general effect on RBP16 RNA binding, however, since methylation conversely increases the association of the protein with mRNA (Goulah and Read 2007). In the case of SFPQ/PSF, it is clear that arginine methylation increases its association with mRNA.…”

Section: Discussionmentioning

confidence: 95%

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA

Snijders

Hautbergue

Bloom

et al. 2015

RNA

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Splicing factor proline-and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associatedsplicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

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“…For glycerol gradient analysis, mitochondrial lysates from PF strain 29-13 (equivalent to 5 ϫ 10 9 cells) were purified as indicated previously (23). Mitochondrial vesicles were lysed in lysis buffer (25 mM Tris (pH 8.0), 50 mM KCl, 10 mM MgOAc, 0.2% Nonidet P-40) for 5 min on ice.…”

Section: Cloning and Expression Of Tbrgg2-thementioning

confidence: 99%

TbRGG2, an Essential RNA Editing Accessory Factor in Two Trypanosoma brucei Life Cycle Stages

Fisk

Ammerman²,

Presnyak

et al. 2008

Journal of Biological Chemistry

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165

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In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 downregulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in African wildlife. During their life cycle, T. brucei are transmitted between two different hosts, the tse tse fly insect vector and mammalian host. Due to the resulting drastic changes in environmental growth conditions, the parasite displays differentiationdependent mechanisms of energy metabolism. In the insect midgut stage (procyclic form (PF) 3 ), energy is generated through cytochrome-mediated oxidative phosphorylation, whereas in the mammalian bloodstream form (BF), energy is generated strictly through glycolysis. Correspondingly, the single mitochondrion of T. brucei undergoes extensive alterations in both morphology and gene expression during differentiation. One aspect of this mitochondrial gene regulation is uridine insertion/deletion RNA editing, a process that is restricted to kinetoplastid protozoa, of which T. brucei is a member. During this process, uridine residues are posttranscriptionally added to and/or deleted from pre-mRNAs to produce translatable mature mRNAs. In T. brucei, the accumulation of many edited RNAs is life cycle stage-dependent, with RNAs encoding the cytochrome components apocytochrome b (CYb) and cytochrome oxidase subunit II (COII) edited only in PF and editing of components of the NADH dehydrogenase complex up-regulated i...

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Differential Effects of Arginine Methylation on RBP16 mRNA Binding, Guide RNA (gRNA) Binding, and gRNA-containing Ribonucleoprotein Complex (gRNP) Formation

Cited by 31 publications

References 50 publications

gRNA/pre-mRNA annealing and RNA chaperone activities of RBP16

gRNA/pre-mRNA annealing and RNA chaperone activities of RBP16

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA

TbRGG2, an Essential RNA Editing Accessory Factor in Two Trypanosoma brucei Life Cycle Stages

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