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. 2008 Oct;28(19):6033-43.
doi: 10.1128/MCB.00726-08. Epub 2008 Jul 21.

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

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Free PMC article

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

Julian P Venables et al. Mol Cell Biol. .
Free PMC article

Abstract

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.

Figures

FIG. 1.
FIG. 1.
Alternative splicing of 56 ASEs in response to knockdown of 14 hnRNPs with two siRNAs. Individual hnRNP knockdowns and ASEs are shown to indicate which knockdowns caused a shift in alternative splicing in various cell lines (B = BJT, H = HeLa, and P = PC-3). The type of splicing events being followed is indicated on the right. Each column represents a distinct knockdown performed with the siRNAs listed in Table 1. Shifts with Z-scores of ≥1.5 or ≤−1.5 are shown as bright red and blue bars, respectively, with shifts with Z-scores of ≥2 or ≤−2 indicated with a darker color and Z-scores of ≥1 or ≤1 indicated with a lighter color. White areas indicate no shifts, and gray areas indicate no expression or failed PCRs or knockdown. ASEs that were significantly shifted by both siRNAs in all three cell lines are boxed in yellow. Also boxed in dashed yellow are examples where lower thresholds suggest an effect in all cell lines. (To access individual experiments, go to http://palace.lgfus.ca/to obtain an interactive version of the same figure. Then click on any bar to consult the electropherograms of RT-PCR products fractionated on a Caliper microfluidic station.)
FIG. 2.
FIG. 2.
Examples of hnRNP-regulated ASEs (hits). Selected units that were affected by hnRNP C (PCBP4 and BCL2L12) and hnRNP K (APAF1 and PTK2B) knockdowns in all cell lines are presented. at the top is a diagram of the alternative splicing unit being monitored. Below each alternative splicing unit are gel-like representations of electropherograms corresponding to control and knockdown (+) samples. The position of the products (s = skipped, i = included) corresponds to the expected sizes.
FIG. 3.
FIG. 3.
Representation of hits for each hnRNP protein in the three cell lines. Venn diagrams are used to indicate the number of significant alternative splicing units for each hnRNP (ASEs with shift |Z|-scores >1.5 with both siRNAs) that were found in BJT, HeLa, and PC-3 cells, as well as the overlap between hits.
FIG. 4.
FIG. 4.
Clustered binary map of significantly shifting alternative splicing units. Binary ΔΨ data for the hnRNP knockdown affecting different ASEs in various cell lines. The cell line in which the shift was observed for a specific ASE is indicated (B = BJT, H = HeLa, and P = PC-3). Red indicates an increase in Ψ (e.g., exon inclusion or use of the more internal splice site in alternative 5′ or 3′ splice sites), and blue indicates a decrease, upon hnRNP knockdown. Only shifts with Z-score of ±1.5 or better obtained with two siRNAs are indicated. Gray bars indicate no change. Incomplete data representing depletions that failed (H and Q in BJT cells) or the absence of any RT-PCR products are shown as white bars.

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