Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.     

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

N⁶-methyladenosine (m⁶A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m⁶A demethylases and cell-type and cell-state-dependent m⁶A patterns indicate that m⁶A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m⁶A modification include mRNA splicing, export, stability, and immune tolerance; but m⁶A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m⁶A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m⁶A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m⁶A modification. We applied the method to determine the m⁶A status at several sites in two human lncRNAs and three human mRNAs and found that m⁶A fraction varies between 6% and 80% among these sites. We also found that many m⁶A candidate sites in these RNAs are however not modified. The precise determination of m⁶A status in a long noncoding RNA also enables the identification of an m⁶A-containing RNA structural motif.     

Genome-wide profiling of the C. elegans dsRNAome

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3′ UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.     

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.     

RBS1, an RNA Binding Protein,Interacts with SPIN1 and Is Involved in Flowering Time Control in Rice

The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.     

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3–6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T_½ = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.