Chapter 27 Cell Biology of mRNA Decay

Grünwald, David; Singer, Robert H.; Czaplinski, Kevin

doi:10.1016/s0076-6879(08)02627-x

Cited by 23 publications

(18 citation statements)

References 47 publications

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“…To directly show the association of collagen mRNAs with vimentin filaments in vivo, we utilized RNA FISH. This method can detect single mRNA molecules in the cytoplasm (4,19,24,31,59). To visualize collagen mRNAs, we designed multiple short 50-mer oligonucleotide probes that specifically hybridize with their target mRNAs.…”

Section: Resultsmentioning

confidence: 99%

A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

Challa

Stefanovic

2011

Molecular and Cellular Biology

144

130

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The stem-loop in the 5 untranslated region (UTR) of collagen ␣1(I) and ␣2(I) mRNAs (5SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5SL of ␣1(I) and ␣2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5SL-and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen ␣1(I) and ␣2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen ␣1(I) and ␣2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies.Fibroproliferative disorders are leading causes of morbidity and mortality globally (1,5,11,20,28,38) and pose an enormous threat to human health. There are no effective therapies for fibrotic diseases. Excessive production of type I collagen by activated fibroblasts and myofibroblasts is the hallmark of fibroproliferative disorders. Type I collagen is a heterotrimer composed of two ␣1(I) chains and one ␣2(I) chain (29). The increased collagen synthesis can be due to the increased rate of transcription of collagen genes, increased half-life of collagen mRNAs, and their enhanced translation (33,35,47,51). Increased collagen production by activated fibroblasts is primarily due to an increase in stability of collagen mRNAs (16). During activation of hepatic stellate cells, which synthesize collagen in liver fibrosis, a 16-fold prolongation of the half-life of collagen ␣1(I) mRNAs is primarily responsible for its 50-fold-increased expression (45). The transformation of fibroblasts into myofibroblasts is also associated with increased stability of collagen ␣1(I) mRNA (37). Transforming growth factor beta (TGF-␤), the most potent profibrotic cytokine, induces collagen synthesis by prolonging the half-life of collagen ␣1(I) mRNA (51). Thus, it is now well established that stability of collagen mRNAs is the predominant mechanism regulating collagen expression (30,40,41).Two cis-acting elements were implicated in regulating the stability of collagen mRNAs. In the 3Ј untranslated region (3Ј UTR) of collagen ␣1(I) mRNA, there is a cytosine-rich sequence that interacts with the ␣CP protein; this interaction stabilizes the mRNA (32, 34). In the 5Ј UTR of col...

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Section: Resultsmentioning

confidence: 99%

A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

Challa

Stefanovic

2011

Molecular and Cellular Biology

144

130

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“…A modified version of Robert Singer's laboratory protocol [23] was used. Oligonucleotide probes (50-nucleotides long) were designed using Vector NTI software (Invitrogen), and the probe specificity was verified with Ensembl BlastView tool.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes1

Flemr

Schultz

et al. 2010

Biology of Reproduction

125

134

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In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

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“…Since the use of extensive washes to remove salts and formamide after the FISH procedure and before IF did not help, we chose the IF->FISH procedure as a more promising variant for further development. Our choice of IF followed by FISH was further supported by the protocol proposed by Grnwald et al (12), which we found partially successful but insufficient in terms of signal intensities, staining patterns, and the formation of secondary antibody artifactual speckles (discussed in detail below).…”

Section: Resultsmentioning

confidence: 55%

Simultaneous Detection of mRNA and Protein in Single Cells Using Immunofluorescence-Combined Single-Molecule RNA FISH

2015

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Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

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Chapter 27 Cell Biology of mRNA Decay

Cited by 23 publications

References 47 publications

A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes1

Simultaneous Detection of mRNA and Protein in Single Cells Using Immunofluorescence-Combined Single-Molecule RNA FISH

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