Light-Activated Control of Translation by Enzymatic Covalent mRNA Labeling.

Activation of cellular protein expression upon visible-light photocleavage of small-molecule caging groups covalently attached to the 5' untranslated region (5' UTR) of an mRNA was achieved. These photocleavable caging groups are conjugated to in vitro transcribed mRNA (IVT-mRNA) through RNA transglycosylation, an enzymatic process in which a bacterial tRNA guanine transglycosylase (TGT) exchanges a guanine nucleobase in a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine1 (preQ1 ) derivatives. The caging groups severely reduce mRNA translation efficiency when strategically placed in the 5' UTR. Using this method, we demonstrate the successful spatiotemporal photoregulation of gene expression with single-cell precision. Our method can be applied to therapeutically relevant chemically modified mRNA (mod-mRNA) transcripts. This strategy provides a modular and efficient approach for developing synthetic gene regulatory circuits, biotechnological applications, and therapeutic discovery.

DNA double-strand breaks relieve USF-mediated repression of Dß2 germline transcription in developing thymocytes.

Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRß locus (Tcrb) D segments, Dß1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJß2 segment cluster is initially restricted to the J segments and only redirected upstream of Dß2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJß2 recombination is less efficient than that of DJß1. Because inefficient DJß2 assembly offers the potential for V-to-DJß2 recombination to rescue frameshifted V-to-DJß1 joints, we wished to determine how Dß2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dß2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dß2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dß2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dß2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dß2 recombinational accessibility during thymocyte development.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (ß-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the ß-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of ß-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.

Expression of mouse igf2 mRNA-binding protein 3 and its implications for the developing central nervous system.

Functional analyses of neural RNA-binding proteins have focused mainly on their roles as modulators of posttranscriptional gene regulation, e.g., alternative splicing, dendritic mRNA localization, and local translation. Here we identified a mouse homologue of human IMP3, which is known to bind to and repress the translation of igf2 leader 3 mRNA. The mouse igf2 mRNA-binding protein 3 (mIMP3) is a member of the zipcode binding protein-1 (ZBP-1) family previously reported in chick fibroblast cells. mIMP3 was expressed in undifferentiated neuroepithelial cells and some postmitotic neurons at early embryonic stages (E10.5--E12.5), and its expression level decreased after the midembryonic stage (E12.5) until birth. The expression profile of mIMP3 is very similar to that of mouse igf2 leader 3 mRNA. In vitro UV cross-linking experiments showed that mIMP3 preferentially bound to igf2 leader 3 mRNA rather than igf2 leader 4 mRNA and did not bind the zipcode region of beta-actin or c-myc mRNA. Furthermore, persistent expression of mIMP3 protein in an undifferentiated P19 cell line revealed that mIMP3 inhibited neuronal differentiation morphologically and immunohistochemically. Taken together, these observations raise the possibility that mIMP3 represses neuronal differentiation through the regulation of igf2 mRNA expression.

Light-associated and processing-dependent protein binding to 5' regions of rbcL mRNA in the chloroplasts of a C4 plant.

In amaranth, a C(4) dicotyledonous plant, the plastid rbcL gene (encoding the large subunit of ribulose-1,5-bisphosphate carboxylase) is regulated post-transcriptionally during many developmental processes, including light-mediated development. To identify post-transcriptional regulators of rbcL expression, three types of analyses (polysome heel printing, gel retardation, and UV cross-linking) were utilized. These approaches revealed that multiple proteins interact with 5' regions of rbcL mRNA in light-grown, but not etiolated, amaranth plants. Light-associated binding of a 47-kDa protein (p47), observed by UV cross-linking, was highly specific for the rbcL 5' RNA. Binding of p47 occurred only with RNAs corresponding to mature processed rbcL transcripts (5'-untranslated region (UTR) terminating at -66); transcripts with longer 5'-UTRs did not associate with p47 in vitro. Variations in the length of the rbcL 5'-UTR were found to occur in vivo, and these different 5' termini may prevent or enhance light-associated p47 binding, possibly affecting rbcL expression as well. p47 binding correlates with light-dependent rbcL polysome association of the fully processed transcripts in photosynthetic leaves and cotyledons but not with cell-specific rbcL mRNA accumulation in bundle sheath and mesophyll chloroplasts.