3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Guo, Yang; Steitz, Joan A.

doi:10.1261/rna.045054.114

Cited by 22 publications

(23 citation statements)

References 18 publications

(28 reference statements)

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“…1c). RNA-5, the 3′-biotinylated analog of RNA-4, was inactive, consistent with a report that biotin modification at the miRNA 3′ terminus may prevent uptake into miRISC 15 . However, RNA-6 and the pre-miRNA analog RNA-7 bearing biotin at internal positions were only slightly less active than unmodified miR-106a and pre-miR-106a, respectively.…”

Section: Mir-clip Capture Of a Mirna Targetome Uncovers A Lincrna H19supporting

confidence: 89%

“…In two recent studies, biotinylated miRNA mimics were used to capture mRNA targets 13,14 . However, these methods have the potential to generate false positives from probetarget interactions outside the miRISC 15 and false negatives owing to the loss of target RNAs during purification 16 . Solutions to these potential shortcomings are immunoprecipitation of the mRNA ribonucleoprotein complexes 17 and crosslinking of the miRNA-RNA partners 13,18 before sample work-up.…”

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confidence: 99%

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miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19–miR-106a interaction

et al. 2014

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Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

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Section: Mir-clip Capture Of a Mirna Targetome Uncovers A Lincrna H19supporting

confidence: 89%

mentioning

confidence: 99%

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19–miR-106a interaction

et al. 2014

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“…RNA oligonucleotides 5′-A 9 GCA 4 -3′ (Dharmacon), 5′-phosphorylated UUCACAGUGGCUAAGUUCCGC-3′ biotin via C6 linker (IDT), and 5′-GGAACUUAGCCACUGUGGAAG-3′ (IDT) were prepared as described (5,27). For EMSA, the MALAT1 (nucleotides 8263-8355), MENβ (nucleotides 22651-22743), and KSHV PAN (nucleotides 895-964) ENEs were transcribed by his-tagged T7 RNA polymerase using a PCR-generated template, and the RNA product was processed as described (5) except that the 5′-triphosphate moiety was removed by using calf intestinal alkaline phosphatase (New England Biolabs) per the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

Brown

Kinzig

DeGregorio

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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211

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; reviewed by Brenda L. Bass and Eric M. Phizicky)Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3′-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U • A-U triples interrupted by a C • G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 10 5 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.RNA binding protein | RNA triple helix | noncoding RNA

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“…Terminal chemical modifications, including Cy3-, cholesterol-, biotin-and amino-modified oligonucleotides, can increase the stability and tracer function of oligonucleotides for in vivo delivery [39,40]. In practical applications, multiple modifications are used together to increase the stability, delivery and cellular uptake efficiency of oligonucleotides in vivo.…”

Section: Therapeutic Approaches Involving Mirnamentioning

confidence: 99%

Recent progress in microRNA-based delivery systems for the treatment of human disease

Chen

Huang

2019

ExRNA

144

118

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MicroRNAs (miRNAs) are naturally occurring, small non-coding RNAs that mediate posttranscriptional regulation. Based on the level of sequence complementarity, miRNAs lead to the degradation of target mRNAs or the suppression of mRNA translation, thereby inhibiting the synthesis of proteins and achieving the regulation of genes. miRNAs, which exhibit tissue-and temporal-specific expression, are important negative regulatory RNAs that decrease the levels of other functional genes. miRNAs play a crucial role in disease progression and prognosis and thus exhibit potential for developing novel therapeutics. Due to the instability of miRNAs and their complex environment, including degradation by nucleases in vivo, the safety and effectiveness of miRNA delivery has become the focus of recent attention. Therefore, we discuss some representative advances related to the application of viral-and nonviral-mediated miRNA delivery systems and provide a new perspective on the future of miRNA-based therapeutic strategies.

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3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Cited by 22 publications

References 18 publications

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19–miR-106a interaction

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19–miR-106a interaction

Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

Recent progress in microRNA-based delivery systems for the treatment of human disease

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