Substrate specificity of Mycobacterium tuberculosis tRNA terminal nucleotidyltransferase toxin MenT3.

Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.

Mechanism of U6 snRNA oligouridylation by human TUT1.

U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA.

Mechanism of tRNA recognition by heterotetrameric glycyl-tRNA synthetase from lactic acid bacteria.

Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2ß2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2ß2 GlyRS, consisting of the full-length α and ß subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2ß2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2ß2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and ß subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and ß subunits of LpGlyRS together interact with the 3'-end and the acceptor region of tRNAGly, and the C-terminal domain of the ß subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors.

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA-CT) into neighboring bacteria. The CdiA-CT of enterohemorrhagic Escherichia coli EC869 (CdiA-CTEC869) cleaves the 3'-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA-CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA-CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA-CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA-CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA-CTEC869 interacts with domain II of Tu, thereby preventing the 3'-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA-CTEC869:Tu structure suggests that the 3'-portion of tRNA relocates into the CdiA-CTEC869 active site, located on the opposite side to the CdiA-CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA-CTEC869 is recruited to Tu:GTP:Ts, and CdiA-CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA-CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA-CTEC869.

Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance.

Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

Molecular basis of glycyl-tRNAGly acetylation by TacT from Salmonella Typhimurium.

Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNAGlyin vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNAGly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNAGly, a combination that is only found in tRNAGly isoacceptors, and discriminates tRNAGly from other tRNA species. Thus, TacT is a Gly-tRNAGly-specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella.

Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli.

Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs.

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.

Mechanistic insights into m6A modification of U6 snRNA by human METTL16.

The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5'-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16-U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

Crystal structure of human cytoplasmic tRNAHis-specific 5'-monomethylphosphate capping enzyme.

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

Crystal structure of the Lin28-interacting module of human terminal uridylyltransferase that regulates let-7 expression.

Lin28-dependent oligo-uridylylation of precursor let-7 (pre-let-7) by terminal uridylyltransferase 4/7 (TUT4/7) represses let-7 expression by blocking Dicer processing, and regulates cell differentiation and proliferation. The interaction between the Lin28:pre-let-7 complex and the N-terminal Lin28-interacting module (LIM) of TUT4/7 is required for pre-let-7 oligo-uridylylation by the C-terminal catalytic module (CM) of TUT4/7. Here, we report crystallographic and biochemical analyses of the LIM of human TUT4. The LIM consists of the N-terminal Cys2His2-type zinc finger (ZF) and the non-catalytic nucleotidyltransferase domain (nc-NTD). The ZF of LIM adopts a distinct structural domain, and its structure is homologous to those of double-stranded RNA binding zinc fingers. The interaction between the ZF and pre-let-7 stabilizes the Lin28:pre-let-7:TUT4 ternary complex, and enhances the oligo-uridylylation reaction by the CM. Thus, the ZF in LIM and the zinc-knuckle in the CM, which interacts with the oligo-uridylylated tail, together facilitate Lin28-dependent pre-let-7 oligo-uridylylation.

Crystal Structure of the Enterohemorrhagic Escherichia coli AtaT-AtaR Toxin-Antitoxin Complex.

AtaT-AtaR is an enterohemorrhagic Escherichia coli toxin-antitoxin system that modulates cellular growth under stress conditions. AtaT and AtaR act as a toxin and its repressor, respectively. AtaT is a member of the GNAT family, and the dimeric AtaT acetylates the α-amino group of the aminoacyl moiety of methionyl initiator tRNAfMet, thereby inhibiting translation initiation. The crystallographic analysis of the AtaT-AtaR complex revealed that the AtaT-AtaR proteins form a heterohexameric [AtaT-(AtaR4)-AtaT] complex, where two V-shaped AtaR dimers bridge two AtaT molecules. The N-terminal region of AtaR is required for its dimerization, and the C-terminal region of AtaR interacts with AtaT. The two AtaT molecules are spatially separated in the AtaT-AtaR complex. AtaT alone forms a dimer in solution, which is enzymatically active. The present structure, in which AtaR prevents AtaT from forming an active dimer, reveals the molecular basis of the AtaT toxicity repression by the antitoxin AtaR.

Function and Regulation of Human Terminal Uridylyltransferases.

RNA uridylylation plays a pivotal role in the biogenesis and metabolism of functional RNAs, and regulates cellular gene expression. RNA uridylylation is catalyzed by a subset of proteins from the non-canonical terminal nucleotidyltransferase family. In human, three proteins (TUT1, TUT4, and TUT7) have been shown to exhibit template-independent uridylylation activity at 3'-end of specific RNAs. TUT1 catalyzes oligo-uridylylation of U6 small nuclear (sn) RNA, which catalyzes mRNA splicing. Oligo-uridylylation of U6 snRNA is required for U6 snRNA maturation, U4/U6-di-snRNP formation, and U6 snRNA recycling during mRNA splicing. TUT4 and TUT7 catalyze mono- or oligo-uridylylation of precursor let-7 (pre-let-7). Let-7 RNA is broadly expressed in somatic cells and regulates cellular proliferation and differentiation. Mono-uridylylation of pre-let-7 by TUT4/7 promotes subsequent Dicer processing to up-regulate let-7 biogenesis. Oligo-uridylylation of pre-let-7 by TUT4/7 is dependent on an RNA-binding protein, Lin28. Oligo-uridylylated pre-let-7 is less responsive to processing by Dicer and degraded by an exonuclease DIS3L2. As a result, let-7 expression is repressed. Uridylylation of pre-let-7 depends on the context of the 3'-region of pre-let-7 and cell type. In this review, we focus on the 3' uridylylation of U6 snRNA and pre-let-7, and describe the current understanding of mechanism of activity and regulation of human TUT1 and TUT4/7, based on their crystal structures that have been recently solved.

Human BCDIN3D Is a Cytoplasmic tRNAHis-Specific 5'-Monophosphate Methyltransferase.

Bicoid interacting 3 domain containing RNA methyltransferase (BCDIN3D) is a member of the Bin3 methyltransferase family and is evolutionary conserved from worm to human. BCDIN3D is overexpressed in breast cancer, which is associated with poor prognosis of breast cancers. However, the biological functions and properties of BCDIN3D have been enigmatic. Recent studies have revealed that human BCDIN3D monomethylates 5'-monophsosphate of cytoplasmic tRNAHisin vivo and in vitro. BCDIN3D recognizes the unique and exceptional structural features of cytoplasmic tRNAHis and discriminates tRNAHis from other cytoplasmic tRNA species. Thus, BCDIN3D is a tRNAHis-specific 5'-monophosphate methyltransferase. Methylation of the 5'-phosphate group of tRNAHis does not significantly affect tRNAHis aminoacylation by histidyl-tRNA synthetase in vitro nor the steady state level or stability of tRNAHisin vivo. Hence, methylation of the 5'-phosphate group of tRNAHis by BCDIN3D or tRNAHis itself may be involved in certain unknown biological processes, beyond protein synthesis. This review discusses recent reports on BCDIN3D and the possible association between 5'-phosphate monomethylation of tRNAHis and the tumorigenic phenotype of breast cancer.

Acetate-dependent tRNA acetylation required for decoding fidelity in protein synthesis.

Modification of tRNA anticodons plays a critical role in ensuring accurate translation. N4-acetylcytidine (ac4C) is present at the anticodon first position (position 34) of bacterial elongator tRNAMet. Herein, we identified Bacillus subtilis ylbM (renamed tmcAL) as a novel gene responsible for ac4C34 formation. Unlike general acetyltransferases that use acetyl-CoA, TmcAL activates an acetate ion to form acetyladenylate and then catalyzes ac4C34 formation through a mechanism similar to tRNA aminoacylation. The crystal structure of TmcAL with an ATP analog reveals the molecular basis of ac4C34 formation. The ΔtmcAL strain displayed a cold-sensitive phenotype and a strong genetic interaction with tilS that encodes the enzyme responsible for synthesizing lysidine (L) at position 34 of tRNAIle to facilitate AUA decoding. Mistranslation of the AUA codon as Met in the ΔtmcAL strain upon tilS repression suggests that ac4C34 modification of tRNAMet and L34 modification of tRNAIle act cooperatively to prevent misdecoding of the AUA codon.

Crystal structures of U6 snRNA-specific terminal uridylyltransferase.

The terminal uridylyltransferase, TUT1, builds or repairs the 3'-oligo-uridylylated tail of U6 snRNA. The 3'-oligo-uridylylated tail is the Lsm-binding site for U4/U6 di-snRNP formation and U6 snRNA recycling for pre-mRNA splicing. Here, we report crystallographic and biochemical analyses of human TUT1, which revealed the mechanisms for the specific uridylylation of the 3'-end of U6 snRNA by TUT1. The O2 and O4 atoms of the UTP base form hydrogen bonds with the conserved His and Asn in the catalytic pocket, respectively, and TUT1 preferentially incorporates UMP onto the 3'-end of RNAs. TUT1 recognizes the entire U6 snRNA molecule by its catalytic domains, N-terminal RNA-recognition motifs and a previously unidentified C-terminal RNA-binding domain. Each domain recognizes specific regions within U6 snRNA, and the recognition is coupled with the domain movements and U6 snRNA structural changes. Hence, TUT1 functions as the U6 snRNA-specific terminal uridylyltransferase required for pre-mRNA splicing.

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.