Biology subjects

Tengo, L.

Publications and source records attributed to Tengo, L..

5 recordsLinked to original sources

bioRxiv2026

The RNA helicase KREH2 directly binds the RESC1-RESC2 complex in trypanosomatid mitochondrial RNA editing

Trypanosomatids are medically important single-celled eukaryotic protists with exceptional RNA biology. They display highly unusual RNA biogenesis pathways, notably mitochondrial RNA editing, an essential post-transcriptional process transforming cryptic primary transcripts into functional mRNAs. Large multiprotein assemblies coordinate the RNA editing process, in which small guide RNAs (gRNAs) anneal to the pre-mRNAs and serve as templates for uridine insertion or deletion. Kinetoplast RNA Editing Helicases (KREHs) are important components of the RNA editing process that modifies large portions of pre-mRNA via the sequential annealing and dissociation of multiple gRNAs. However, how KREH helicases interact with components of the editing machinery, and which subunits mediate these interactions, remain unresolved. Here, we examined an association between the DEAH/RHA RNA helicase KREH2 and the RESC1-RESC2 protein heterodimer using proximity labeling and co-immunoprecipitation coupled to quantitative mass spectrometry in vivo. We reconstituted a minimal trimeric KREH2 peptide-RESC1-RESC2 complex in vitro and characterized novel direct binding of a KREH2 peptide to RESC1 using cryo-electron microscopy. These results establish a mechanistic link between the RNA helicase KREH2 and the RESC1-RESC2 module at the 5'-end of gRNAs, providing new avenues for studying the orchestration of RNA remodeling in trypanosomatid mitochondrial RNA editing.

molecular biology↗

bioRxiv2026

The C-terminus of the KREH1 helicase is important for RNA binding and association with mitochondrial RNA editing complexes in T. brucei

Mitochondrial pre-mRNA editing in kinetoplastids, a clade that includes human-infective parasitic protists such as Trypanosoma and Leishmania, is required to produce translatable, functional gene products from otherwise nonfunctional mitochondrial precursor transcripts of essential respiratory chain complex subunits. RNA editing is a template-guided process in which cognate guide RNAs (gRNAs) anneal to specific sites at the pre-mRNA and direct the complementary insertion and/or deletion of uridines. Large multi-modular complexes - like the RNA-Editing Substrate-binding Complex (RESC) and the RNA-Editing Catalytic Complexes (RECC) - organize and catalyze RNA editing, respectively. Within these assemblies, Kinetoplast RNA Editing Helicases (KREHs) play an important role in ribonucleoprotein complex remodeling, particularly in pan-editing, when transcripts are extensively modified throughout their length by multiple gRNAs. However, the mechanisms by which KREH RNA helicases facilitate pan-editing, and their specific roles in this process, remain unclear. Here, we define the molecular properties of the Trypanosoma brucei DEAD-box RNA editing helicase KREH1. Cellular co-proteome analyses reveal that KREH1 interacts with subunits of the RESC complex and biochemical experiments show that KREH1 is an RNA-dependent ATPase that unwinds double-stranded RNA substrates. We show that the flexible C-terminus of KREH1 is important for RNA binding and, consequently, for enzymatic activity in vitro and interactions with the RESC complex in vivo. Overall, our experiments refine the role of KREH1 in the pan-editing process and establish its utility as a molecular tool for investigating higher-order RNA editing complexes in the cellular context.

molecular biology↗

bioRxiv2024

Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex

Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5-end of each mRNA. The 5-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development. HighlightsO_LICryo-EM reveals the molecular architecture of the tetrameric Trypanosoma brucei cap-binding complex (TbCBC). C_LIO_LITbCBP110 is the kinetoplastid homolog of mammalian CBP80 and forms the scaffold for TbCBP20. C_LIO_LITbCBC has a bilobal architecture with TbCBP30 bridging the flexibly attached TbCBP66 subunit and the TbCBP20-TbCBP110 core complex. C_LIO_LITbCBC recognizes the m7G RNA cap independent of the other trypanosomatid-specific cap4 methylations. C_LIO_LIThe TbCBP66 subunit contains a binding site for dsRNA, augmenting the affinity of TbCBC for the SL RNA. C_LI

molecular biology↗

bioRxiv2023

Structural basis of tRNA recognition by the m3C-RNA-methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity 1. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances 2,3. However, our understanding of the substrate selection and catalysis mode of the m3C RNA methyltransferases is currently still lacking. Here, we report the cryo-electron microscopy structure of the m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We reveal how METTL6 and SerRS jointly coordinate the long variable arm of tRNASer in their interface. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, based on the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA binding mode for m3C RNA methyltransferases including METTL2 and METTL8, suggesting that these mammalian paralogues use similar ways to engage their respective tRNA substrates and co-factors.

molecular biology↗

bioRxiv2022

Sequence-independent substrate selection by the eukaryotic wobble base deaminase ADAT2/3 involves multiple protein domains and distortion of the tRNA anticodon loop

The essential deamination of adenosine A34 to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the first structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A novel gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.

molecular biology↗