Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

Trask, Heidi W.; Cowper-Sal·lari, Richard; Sartor, Maureen A.; Gui, Jiang; Heath, Catherine V.; Renuka, Janhavi; Higgins, Azara Jane; Andrews, Peter C.; Korc, Murray; Moore, Jason H.; Tomlinson, Craig R.

doi:10.1261/rna.1677409

Cited by 30 publications

(47 citation statements)

References 40 publications

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“…Despite the small number of the analyzed RNAs, these results suggest that the AGCCC motif may play a role in nuclear localization of additional lncRNAs and proteincoding RNAs. It is known that a significant fraction of the transcript population of many protein-coding RNAs is nucleus-localized (66,67), and at least in some cases, nuclear retention is used as a means for regulation of the level of translation (68,69). In addition, increasing evidence suggests that the protein-coding RNAs have functions beyond merely coding for proteins, and indeed, many of them may regulate cellular processes as functional RNAs (70).…”

Section: Discussionmentioning

confidence: 99%

A Novel RNA Motif Mediates the Strict Nuclear Localization of a Long Noncoding RNA

Zhang

Gunawardane

Niazi

et al. 2014

Molecular and Cellular Biology

132

113

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The ubiquitous presence of long noncoding RNAs (lncRNAs) in eukaryotes points to the importance of understanding how their sequences impact function. As many lncRNAs regulate nuclear events and thus must localize to nuclei, we analyzed the sequence requirements for nuclear localization in an intergenic lncRNA named BORG (BMP2-OP1-responsive gene), which is both spliced and polyadenylated but is strictly localized in nuclei. Subcellular localization of BORG was not dependent on the context or level of its expression or decay but rather depended on the sequence of the mature, spliced transcript. Mutational analyses indicated that nuclear localization of BORG was mediated through a novel RNA motif consisting of the pentamer sequence AGCCC with sequence restrictions at positions ؊8 (T or A) and ؊3 (G or C) relative to the first nucleotide of the pentamer. Mutation of the motif to a scrambled sequence resulted in complete loss of nuclear localization, while addition of even a single copy of the motif to a cytoplasmically localized RNA was sufficient to impart nuclear localization. Further, the presence of this motif in other cellular RNAs showed a direct correlation with nuclear localization, suggesting that the motif may act as a general nuclear localization signal for cellular RNAs.

show abstract

Section: Discussionmentioning

confidence: 99%

A Novel RNA Motif Mediates the Strict Nuclear Localization of a Long Noncoding RNA

Zhang

Gunawardane

Niazi

et al. 2014

Molecular and Cellular Biology

132

113

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“…Nuclear RNA was obtained from nuclear extracts by the same method (Trask et al 2009). Polyadenylated RNA was isolated from 1 mg total RNA with an mRNA Purification Kit (GE Healthcare).…”

Section: Rna Analysismentioning

confidence: 99%

“…Nuclear extract (Trask et al 2009) from siRNA-treated U2OS cells was loaded on a linear 10%-45% sucrose gradient (150 mM NaCl, 10 mM HEPES pH 7.6, 1.5 mM MgCl 2 ). After centrifugation in a SW40Ti rotor (Beckman) at 40,000 rpm and 4°C for 4 h, 18 fractions were collected.…”

Section: Gradient Analysismentioning

confidence: 99%

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Berndt¹,

Harnisch²,

Rammelt³

et al. 2012

RNA

134

171

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Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 39 end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 39 end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 39 end trimming, which might serve to enhance snoRNA stability.

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“…Other factors can also contribute to the limited overlap between the two sets of data, including the differences inherent to each technique and the use of a different starting material for microarray and deep sequencing (cytoplasmic vs. total extracts, respectively). Large differences have been reported in a recent microarray analysis comparing whole-cell versus cytoplasmic RNA (Trask et al 2009). These investigators found that up to 31% of the differentially expressed genes detected using cytoplasmic RNA can be missed by using total RNA and that the contribution of the nuclear RNA to total RNA variation is far from negligible.…”

Section: Discussionmentioning

confidence: 98%

“…These data indicate that the variation between independent biological replicates is relatively small and thus should contribute little to the limited overlap between the RIP-Chip and RIP-seq data. Other factors such as the use of total versus cytoplasmic RNA have the potential to contribute more decisively, because large variations have been recently reported when these two types of starting material are compared in gene expression studies using microarrays (Trask et al 2009) (see Discussion).…”

Section: à6mentioning

confidence: 99%

Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR

Mihailovich

Wurth

Zambelli

et al. 2011

RNA

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Upstream of N-ras (UNR) is a conserved RNA-binding protein that regulates mRNA translation and stability by binding to sites generally located in untranslated regions (UTRs). In Drosophila, sex-specific binding of UNR to msl2 mRNA and the noncoding RNA roX is believed to play key roles in the control of X-chromosome dosage compensation in both sexes. To investigate broader sex-specific functions of UNR, we have identified its RNA targets in adult male and female flies by high-throughput RNA binding and transcriptome analysis. Here we show that UNR binds to a large set of protein-coding transcripts and to a smaller set of noncoding RNAs in a sex-specific fashion. The analyses also reveal a strong correlation between sex-specific binding of UNR and sex-specific differential expression of UTRs in target genes. Validation experiments indicate that UNR indeed recognizes sex-specifically processed transcripts. These results suggest that UNR exploits the transcript diversity generated by alternative processing and alternative promoter usage to bind and regulate target genes in a sex-specific manner.

show abstract

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

Cited by 30 publications

References 40 publications

A Novel RNA Motif Mediates the Strict Nuclear Localization of a Long Noncoding RNA

A Novel RNA Motif Mediates the Strict Nuclear Localization of a Long Noncoding RNA

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR

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