Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2′-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5′-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A–U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch.     

An innate twist between Crick’s wobble and Watson-Crick base pairs

Non-Watson-Crick pairs like the G·U wobble are frequent in RNA duplexes. Their geometric dissimilarity (nonisostericity) with the Watson-Crick base pairs and among themselves imparts structural variations decisive for biological functions. Through a novel circular representation of base pairs, a simple and general metric scheme for quantification of base-pair nonisostericity, in terms of residual twist and radial difference that can also envisage its mechanistic effect, is proposed. The scheme is exemplified by G·U and U·G wobble pairs, and their predicable local effects on helical twist angle are validated by MD simulations. New insights into a possible rationale for contextual occurrence of G·U and other non-WC pairs, as well as the influence of a G·U pair on other non-Watson-Crick pair neighborhood and RNA-protein interactions are obtained from analysis of crystal structure data. A few instances of RNA-protein interactions along the major groove are documented in addition to the well-recognized interaction of the G·U pair along the minor groove. The nonisostericity-mediated influence of wobble pairs for facilitating helical packing through long-range interactions in ribosomal RNAs is also reviewed.     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.     

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.     

Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.     

RNA editing in trypanosomes

Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4–14 nucleotide anchor sequence embedded in the 5 region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5–24 nucleotide 3 terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3 U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.Abbreviations gRNA guide RNA     

TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome

RNA editing produces mature trypanosome mitochondrial mRNAs by uridylate (U) insertion and deletion. In insertion editing, Us are added to the pre-mRNA by a 3' terminal uridylyl transferase (TUTase) activity. We report the identification of a TUTase activity that copurifies with in vitro editing and is catalyzed by the integral editosome protein TbMP57. TbMP57 catalyzes the addition of primarily a single U to single-stranded (ss) RNA and adds the number of Us specified by a guide RNA to insertion editing-like substrates. TbMP57 is distinct from a previously identified TUTase that adds many Us to ssRNA and which we find is neither a stable editosome component nor does it add Us to editing-like substrates. Recombinant TbMP57 specifically interacts with the editosome protein TbMP81, and this interaction enhances the TUTase activity. These results suggest that TbMP57 catalyzes U addition to pre-mRNA during editing.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

The electrostatic characteristics of G.U wobble base pairs

G·U wobble base pairs are the most common and highly conserved non-Watson–Crick base pairs in RNA. Previous surface maps imply uniformly negative electrostatic potential at the major groove of G·U wobble base pairs embedded in RNA helices, suitable for entrapment of cationic ligands. In this work, we have used a Poisson–Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G·U wobble displays distinctly enhanced negativity compared with standard GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G·U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5kT/e, depending upon structural features. We propose that the negativity at the major groove of G·U wobble base pairs is determined by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G·U sites in RNA are most likely to bind cationic ligands.     

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

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.     

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.     

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistant with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events.     

Uridylate-specific 3' 5'-exoribonucleases involved in uridylate-deletion RNA editing in trypanosomatid mitochondria

In kinetoplastid protists, maturation of mitochondrial pre-mRNAs involves the insertion and deletion of uridylates (Us) within coding regions, as specified by mitochondrial DNA-encoded guide RNAs. U-deletion editing involves endonucleolytic cleavage of the pre-mRNA at the editing site followed by U-specific 3'-5'-exonucleolytic removal of nonbase-paired Us prior to ligation of the two mRNA cleavage fragments. We showed previously that an exonuclease/endonuclease/phosphatase (EEP) motif protein from Leishmania major, designated RNA editing exonuclease 1 (REX1) (Kang, X., Rogers, K., Gao, G., Falick, A. M., Zhou, S.-L., and Simpson, L. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1017-1022), exhibits 3'-5'-exonuclease activity. Two EEP motif proteins have also been identified in the Trypanosoma brucei editing complex. TbREX1 is a homologue of LmREX1, and TbREX2 shows homology to another editing protein in L. major, which lacks the EEP motif (LmREX2*). Here we have expressed the T. brucei EEP motif proteins in insect cells and purified them to homogeneity. We showed that these are U-specific 3'-5'-exonucleases that are inhibited by base pairing of 3' Us. The recombinant EEP motif alone also showed 3'-5' U-specific exonuclease activity, and mutations of the REX EEP motifs greatly reduced exonuclease activity. The absence of enzymatic activity in LmREX2* was confirmed with a purified recombinant protein. We showed that pre-cleaved U-deletion editing could be reconstituted with either TbREX1 or TbREX2 in combination with either RNA ligase, LmREL1, or LmREL2. Down-regulation of TbREX2 expression by conditional RNA interference had little effect on parasite viability or sedimentation of the L-complex, suggesting either that TbREX2 is inactive in vivo or that TbREX1 can compensate for the loss of TbREX2 function in down-regulated cells.