Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics.
Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics.
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DOI：
10.1002/cbic.201200091
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发表时间：
2012-05-29
期刊：
影响因子：
3.2
通讯作者：
Conrad, Nicholas K.
中科院分区：
文献类型：
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作者：
Grammel, Markus;Hang, Howard;Conrad, Nicholas K.
Understanding the mechanisms of gene expression requires a thorough knowledge of the balance between the biogenesis of an mRNA and its degradation. While advances in transcriptome profiling methods have enabled the large-scale analysis of RNA steady-state levels, monitoring the synthesis, processing and decay rates of specific transcripts remains technically challenging. The application of nucleoside analogs such as 4-thiouridine (4-sU) has recently enabled metabolic labeling and enrichment of nascent transcripts to monitor RNA dynamics in mammalian cells.[1] Thiol-selective labeling allows the capture and enrichment of RNA but is incompatible with cellular imaging due to competing free thiols in cells. Alternatively, metabolic labeling with 5-ethynyluridine (EU) is compatible with fluorescence imaging of nascent transcripts in cells, tissues and model organisms following copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or “click chemistry”) with azidefunctionalized fluorescent dyes.[2] Here we report N6-propargyl adenosine [3](N6pA; Figure 1a) is efficiently incorporated into RNA in mammalian cells by polyadenylate (poly (A)) polymerase and all three mammalian RNA polymerases: pol I, which transcribes ribosomal RNA in the nucleolus, pol II which transcribes mRNA and some noncoding RNAs, and pol III which transcribes specific small RNAs (tRNAs, et al.). We show that N6pA can be used for CuAAC-mediated fluorescence imaging and affinity enrichment of nascent transcripts. Finally, we demonstrate how “clickable” nucleosides can be employed to monitor poly (A) tail dynamics in cells.To determine whether N6pA labels cellular RNA, we analyzed metabolic incorporation of N6pA into mammalian cells by fluorescence microscopy.[2] HeLa cells were incubated in complete medium containing a range of N6pA concentrations (1.0 µM-1 mM). After 12 hours, the cells were washed, permeabilized, fixed, reacted with azido-rhodamine via CuAAC and visualized by fluorescence microscopy (Figure 1b). The majority of N6pA-specific fluorescence signal localized to distinct substructures of the nucleus, most likely nucleoli, in accordance with the dominant transcriptional activity of pol I. Incorporation of N6pA was observed with as little as 10 µM of N6pA and appeared to reach its maximum between 10 µM and 100 µM. To distinguish between DNA and RNA labeling, we examined
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DOI：
10.1006/meth.1998.0705
发表时间：
1999-01-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Sallés, FJ;Richards, WG;Strickland, S
通讯作者：
Strickland, S
影响因子：
3.5
作者：
Apponi, Luciano H.;Leung, Sara W.;Pavlath, Grace K.
通讯作者：
Pavlath, Grace K.
影响因子：
15
作者：
Grammel, Markus;Phi Luong;Orth, Kim;Hang, Howard C.
通讯作者：
Hang, Howard C.
DOI：
10.1073/pnas.0808480105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Jao, Cindy Y.;Salic, Adrian
通讯作者：
Salic, Adrian
影响因子：
46.9
作者：
通讯作者：
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