Functional specificity of shuttling hnRNPs revealed by genome-wide analysis of their RNA binding profiles

Abstract

Nab2, Npl3, and Nab4/Hrp1 are essential RNA binding proteins of the shuttling hnRNP class that are required for the efficient export of mRNA. To characterize the in vivo transcript specificity of these proteins, we identified their mRNA binding partners using a microarray-based assay. Each of the three proteins was coimmunoprecipitated with many different mRNA transcripts. Interestingly, each protein exhibits preferential associations with a distinct set of mRNAs. Notably, some of these appear to denote specific functional classes. For example, the ribosomal protein mRNAs and other highly expressed transcripts significantly favor association with Npl3 over Nab2, and Nab4/Hrp1 is strongly enriched with transcripts required for amino acid metabolism. Significantly, nab4 mutants showed a striking, desensitized growth phenotype when exposed to amino acid stress conditions suggesting a biological consequence to the associations we observed. Supporting the hypothesis that these proteins display transcript specificity, we identified a unique 7-nucleotide sequence overrepresented in the transcripts highly associated with Nab2 and Nab4/Hrp1 using the REDUCE algorithm. Validating our approach, our bioinformatics analysis correctly identified the known binding site for Nab4/Hrp1. These specialized associations of the hnRNP proteins of Saccharomyces cerevisiae suggest the opportunity to regulate the processing of particular transcripts between transcription and translation.

FIGURE 1.

Nab2, Npl3, and Nab4/Hrp1 have shared and unique domains. Schematic representation highlighting the known domains of Nab2, Npl3, and Nab4/Hrp1. (Q3P) glutamine and proline-rich region, (RGG) arginine and glycine-rich region, (C3H) zinc finger region, (APQE) alanine, proline, glutamine, glutamic acid-rich region, (RRM) RNA recognition motif.

FIGURE 2.

Each protein has a unique RNA binding spectrum. Graphical representation made with Cluster and Treeview (Eisen et al. 1998) of three replicates for each of the three proteins studied, Nab2, Npl3, and Nab4/Hrp1, using the RNA-IP microarray assay. Each line along the Y-axis represents one gene. Only features that gave reliable signal for every replicate for every protein were included. The data were median centered and normalized, then subjected to SOM and average hierarchical clustering in the Cluster program. Brighter red indicates greater association of that transcript for an hnRNP; brighter green indicates less association.

FIGURE 3.

RPGs are skewed towards association with Npl3 over Nab2. Histogram displaying the distribution of transcripts in the Nab2 versus Npl3 direct IP versus IP microarray experiment relative to the transcript’s ratio on the array. Greater distance to the right of the histograms indicates greater association with Npl3 and vice versa. The distribution of the RPGs are normalized and displayed in gray. (RPGs) ribosomal protein genes.

FIGURE 4.

Mean abundance of Nab2 and Npl3 enriched targets. The average expression estimates (in copies/cell) based upon the Nab2 IP versus Npl3 IP microarray experiments. Estimates were drawn from Wang et al. (2002). (RPGs) Ribosomal protein genes.

FIGURE 5.

Extended motifs identified for Nab2 and Nab4/Hrp1. Sequence logo display of the extended motifs identified by MEME analysis of the highest scoring messages containing the motif generated using the Weblogo tool. The height of the letters indicates the relative frequency of that letter at that position and the overall height of the stack indicates the sequence conservation in terms of information content in bits.

FIGURE 6.

nab4 mutant strains are resistant to branched-chain amino acid stress. Growth assay where a lawn of cells are treated with drops of SMM at the quantities indicated in the lower right panel. SMM specifically inhibits acetolactate synthase that is required for the production of branched-chain amino acids. The zone of growth inhibition (halo) is proportional to the resistance of the strain to the drug. (SMM) sulfometuron methyl.