Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors

Abstract

Background: Pre-mRNA splicing is an essential step in gene expression that occurs co-transcriptionally in the cell nucleus, involving a large number of RNA binding protein splicing factors, in addition to core spliceosome components. Several of these proteins are required for the recognition of intronic sequence elements, transiently associating with the primary transcript during splicing. Some protein splicing factors, such as the U2 small nuclear RNP auxiliary factor (U2AF), are known to be exported to the cytoplasm, despite being implicated solely in nuclear functions. This observation raises the question of whether U2AF associates with mature mRNA-ribonucleoprotein particles in transit to the cytoplasm, participating in additional cellular functions.

Results: Here we report the identification of RNAs immunoprecipitated by a monoclonal antibody specific for the U2AF 65 kDa subunit (U2AF65) and demonstrate its association with spliced mRNAs. For comparison, we analyzed mRNAs associated with the polypyrimidine tract binding protein (PTB), a splicing factor that also binds to intronic pyrimidine-rich sequences but additionally participates in mRNA localization, stability, and translation. Our results show that 10% of cellular mRNAs expressed in HeLa cells associate differentially with U2AF65 and PTB. Among U2AF65-associated mRNAs there is a predominance of transcription factors and cell cycle regulators, whereas PTB-associated transcripts are enriched in mRNA species that encode proteins implicated in intracellular transport, vesicle trafficking, and apoptosis.

Conclusion: Our results show that U2AF65 associates with specific subsets of spliced mRNAs, strongly suggesting that it is involved in novel cellular functions in addition to splicing.

Figure 1

Subcellular distribution of U2AF⁶⁵. (a) RT-PCR analysis of spliced and unspliced β-actin mRNA in cytoplasmic (Cyt) and nuclear (Nuc) fractions isolated from HeLa cells. Primer sequences on actin RNA and size of expected amplification products are depicted below the gel. (b) Western blot analysis of the cytoplasmic (Cyt) and nuclear (Nuc) fractions using antibodies against the indicated proteins. Molecular weight markers are shown on the left. Coomassie staining of the gel used for blotting confirms that both fractions contain similar amounts of total protein. (c) Agarose gel electrophoresis of total RNA extracted after Nycodenz gradient fractionation of cytoplasmic samples. rRNA bands are indicated on the right. Numbers indicate gradient fractions from low to high density. (d) Semiquantitative RT-PCR analysis of actin mRNA in the gradient fractions. (e) Western blot analysis of gradient fractions using anti-U2AF⁶⁵ and anti-PTB antibodies. Molecular weight markers are indicated on the left. The 65 kDa band in the membrane after reprobing for PTB corresponds to residual signal from U2AF⁶⁵ detection. (f) Western blot analysis of gradient fractions and input samples using anti-U2AF⁶⁵ antibody. Extracts were either mock-treated (control) or incubated with RNase A before fractionation. Molecular weight markers are indicated on the left. Arrow points to the intermediate density fraction where U2AF⁶⁵-containing complexes accumulate. PTB, polypyrimidine tract binding protein; RT-PCR, reverse transcription polymerase chain reaction; U2AF, U2 small nuclear RNP auxiliary factor.

Figure 2

Clustering analysis of microarray data. (a) Unsupervised clustering of the microarray dataset was performed with the dChip software using standard settings considering all nonredundant probes with positive hybridization signal. The dataset includes microarray hybridization results from input and immunoprecipitation (IP) samples from three experiments with anti-U2AF⁶⁵ antibody (U1 to U3) and two experiments with anti-PTB antibody (P1 and P2). Sample clustering defines a tree with two first level branches corresponding to input and IP samples. (b) Re-clustering analysis after clearing transcripts that were over-represented either in the inputs or in all immunoprecipitation samples. Sample clustering defines a tree with three first level branches corresponding to input, U2AF⁶⁵, and PTB immunoprecipitation samples. For clustering analysis, the probe signal intensities for each mRNA are standardized to have mean 0 and standard deviation 1 across all samples. The color scale for mRNAs is presented as follows: red represents expression level above mean expression of a gene across all samples, black represents mean expression; and green represents expression lower than the mean. Because of the standardization, probe signal intensities most likely fall within [-3, 3]. PTB, polypyrimidine tract binding protein; U2AF, U2 small nuclear RNP auxiliary factor.

Figure 3

Validation of the mRNA-protein associations identified by microarray analysis. (a) RT-PCR amplification of putative U2AF⁶⁵ associated mRNAs and a nonassociated mRNA (glucose-6-phosphate dehydrogenase [G6PD]) from immunoprecipitation (RIP), mock-imunoprecipitation (Mock), and input samples. For each target, the primer localization and expected size of amplified spliced products is indicated. (b) Comparison of microarray quantification and independent real time-PCR quantification of the enrichment index (log2 immunoprecipitation/input) of selected target mRNAs from anti-U2AF⁶⁵ or anti-PTB immunoprecipitation experiments. Points falling in the positive (+/+) or negative (-/-) sectors of the plot reveal an agreement between microarray and quantitative real-time PCR experiments. The SMARCA2 and MAPK8 mRNA were found to be enriched only in microarray experiments. IP, immunoprecipitation; PTB, polypyrimidine tract binding protein; RT-PCR, reverse transcription polymerase chain reaction; U2AF, U2 small nuclear RNP auxiliary factor.

Figure 4

U2AF⁶⁵ associates with fully spliced mRNAs. Agarose gel electrophoresis of RT-PCR reactions designed to check the splicing status of mRNAs present in control (Mock) and anti-U2AF⁶⁵ (U2AF) RNA-immunoprecipitations. For each target, the transcript exon/intron structure is represented to scale and primer localization and expected size of fully spliced products are indicated. IP, immunoprecipitation; RT-PCR, reverse transcription polymerase chain reaction; U2AF, U2 small nuclear RNP auxiliary factor.

Figure 5

Size analysis of coding sequence and UTRs of U2AF⁶⁵- and PTB-associated mRNA populations. (a) Average size of 5' and 3' UTRs and coding sequence (CDS) for mRNAs in the U2AF⁶⁵-associated or PTB-associated populations and their respective control (nonassociated) populations (Additional data file 2). For this analysis, information for the longest curated transcript available in EMBL [40], GenBank [41], and RefSeq [42] databases was retrieved for all entries in each population, when available (Additional data file 4). Statistically significant differences between the associated and the respective control populations are indicated. (b) Analysis of putative U2AF⁶⁵ and PTB binding motifs in selected mRNA populations. The longest curated transcripts for mRNA accessions in the U2AF⁶⁵-associated or PTB-associated populations and their respective control (nonassociated) populations were searched for putative U2AF⁶⁵- and PTB-binding motifs. Graphs present average U2AF⁶⁵ motif density in the U2AF⁶⁵-associated and control populations, and average PTB motif density in the PTB-associated and control populations. The ratio between values for each associated/control pair is shown. (c) Analysis of the distribution of putative U2AF⁶⁵- and PTB-binding motifs in the different transcript regions. Graphs present average U2AF⁶⁵ motif density by transcript region in the U2AF⁶⁵-associated and control populations and average PTB motif density by transcript region in the PTB-associated and control populations. The ratio between values for each associated/control pair is shown. *P << 0.001. n, population size; n.s., not significant; PTB, polypyrimidine tract binding protein; U2AF, U2 small nuclear RNP auxiliary factor; UTR, untranslated region.