Tagging and Capturing of Lentiviral Vectors Using Short RNAs

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

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A Novel RNA Binding Domain in Tetrahymena Telomerase p65 Initiates Hierarchical Assembly of Telomerase Holoenzyme

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2029-2036.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2029-2036 ◽

Cited By ~ 46

Author(s):

Catherine M. O'Connor ◽

Kathleen Collins

Keyword(s):

Rna Binding ◽

Binding Domain ◽

Cooperative Binding ◽

Hierarchical Assembly ◽

Terminal Domain ◽

Recognition Specificity ◽

Central Stem ◽

Rna Binding Domain ◽

Necessary And Sufficient

ABSTRACT Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) assemble as part of a holoenzyme that synthesizes telomeric repeats at chromosome ends. Genetic approaches have identified proteins that are required for in vivo association of TERT and TER, including the Tetrahymena telomerase holoenzyme protein p65. Here, we use quantitative assays to define the mechanisms underlying p65 function in holoenzyme biogenesis. We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. Finally, we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding. These findings elucidate an extensive network of p65-TER recognition specificity and define a novel p65 RNA binding domain that initiates telomerase holoenyzme biogenesis.

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The RNA Binding Domain of Jerky Consists of Tandemly Arranged Helix-Turn-Helix/Homeodomain-Like Motifs and Binds Specific Sets of mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4083-4093.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4083-4093 ◽

Cited By ~ 18

Author(s):

Wencheng Liu ◽

Jeremy Seto ◽

Etienne Sibille ◽

Miklos Toth

Keyword(s):

Dna Binding ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Binding Domain ◽

Cellular Stress Response ◽

Rna Binding Domain ◽

Necessary And Sufficient ◽

Mrna Binding

ABSTRACT A deficit in the Jerky protein in mice causes recurrent seizures reminiscent of temporal lobe epilepsy. Jerky is present in mRNA particles in neurons. We show that the N-terminal 168 amino acids of Jerky are necessary and sufficient for mRNA binding. The binding domain is similar to the two tandemly arranged homeodomain-like helix-turn-helix DNA binding motifs of centromere binding protein B. The putative helix-turn-helix motifs of Jerky can also bind double-stranded DNA and represent a novel mammalian RNA/DNA binding domain. Microarray analysis identified mRNAs encoding proteins involved in ribosome assembly and cellular stress response that specifically bound to the RNA binding domain of Jerky both in vitro and in vivo. These data suggest that epileptogenesis in Jerky-deficient mice most likely involves pathways associated with ribosome biogenesis and neuronal survival and/or apoptosis.

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The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo

Molecular Microbiology ◽

10.1046/j.1365-2958.1999.01553.x ◽

2002 ◽

Vol 33 (6) ◽

pp. 1118-1131 ◽

Cited By ~ 37

Author(s):

Brian H. Johnstone ◽

Aaron A. Handler ◽

Diana K. Chao ◽

Vi Nguyen ◽

Michael Smith ◽

...

Keyword(s):

G Protein ◽

Rna Binding ◽

Binding Domain ◽

Rna Binding Domain

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Structure of the RBD-PRDI fragment of the antiterminator protein GlcT

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309112020635 ◽

2012 ◽

Vol 68 (7) ◽

pp. 751-756 ◽

Cited By ~ 2

Author(s):

Sebastian Himmel ◽

Christian Grosse ◽

Sebastian Wolff ◽

Claudia Schwiegk ◽

Stefan Becker

Keyword(s):

Bacillus Subtilis ◽

Glucose Metabolism ◽

Rna Binding ◽

Hydrophobic Interactions ◽

Binding Domain ◽

Regulatory Domain ◽

Asymmetric Unit ◽

Rna Sequences ◽

Rna Binding Domain

GlcT is a transcriptional antiterminator protein that is involved in regulation of glucose metabolism inBacillus subtilis. Antiterminator proteins bind specific RNA sequences, thus preventing the formation of overlapping terminator stem-loops. The structure of a fragment (residues 3–170) comprising the RNA-binding domain (RBD) and the first regulatory domain (PRDI) of GlcT was solved at 2.0 Å resolution with one molecule in the asymmetric unit. The two domains are connected by a helical linker. Their interface is mostly constituted by hydrophobic interactions.

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LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain

Nucleic Acids Research ◽

10.1093/nar/gkaa652 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9262-9272 ◽

Cited By ~ 1

Author(s):

Deqiang Ding ◽

Chao Wei ◽

Kunzhe Dong ◽

Jiali Liu ◽

Alexander Stanton ◽

...

Keyword(s):

Rna Binding ◽

Specific Interaction ◽

Binding Activity ◽

Binding Domain ◽

Binding Property ◽

Domain Family ◽

Rna Sequences ◽

G Quadruplex ◽

Rna Binding Domain ◽

Rna Interaction

Abstract LOTUS domains are helix-turn-helix protein folds identified in essential germline proteins and are conserved in prokaryotes and eukaryotes. Despite originally predicted as an RNA binding domain, its molecular binding activity towards RNA and protein is controversial. In particular, the most conserved binding property for the LOTUS domain family remains unknown. Here, we uncovered an unexpected specific interaction of LOTUS domains with G-rich RNA sequences. Intriguingly, LOTUS domains exhibit high affinity to RNA G-quadruplex tertiary structures implicated in diverse cellular processes including piRNA biogenesis. This novel LOTUS domain-RNA interaction is conserved in bacteria, plants and animals, comprising the most ancient binding feature of the LOTUS domain family. By contrast, LOTUS domains do not preferentially interact with DNA G-quadruplexes. We further show that a subset of LOTUS domains display both RNA and protein binding activities. These findings identify the LOTUS domain as a specialized RNA binding domain across phyla and underscore the molecular mechanism underlying the function of LOTUS domain-containing proteins in RNA metabolism and regulation.

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A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0138 ◽

2002 ◽

Vol 13 (10) ◽

pp. 3683-3695 ◽

Cited By ~ 6

Author(s):

Petra Björk ◽

Göran Baurén ◽

ShaoBo Jin ◽

Yong-Guang Tong ◽

Thomas R. Bürglin ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Rna Binding ◽

Ribosomal Subunit ◽

Binding Domain ◽

Dependent Manner ◽

Binding Domains ◽

40S Ribosomal Subunit ◽

Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

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