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.

Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1

“…1d). That Npl3 rather binds to bulk mRNAs was also published earlier 26 . Furthermore, in support of our data, a preferential association of Gbp2 with unspliced transcripts was recently also identified by a transcriptome-wide technique termed CRAC (crosslinking and analysis of cDNA) 27 .…”

“…Together, all data indicate a model in which Npl3 associates with bulk mRNAs, recruited by RNA polymerase II 21,26 . In case of the intron-containing transcripts it participates in the recruitment of the early spliceosome 22 .…”

“…16) and those mature mRNPs are exported into the cytoplasm. Npl3 associates with bulk mRNAs and is recruited early by RNA polymerase II 21,26 and might signal by recruitment of Mex67 that early processing events such as 5 0 -capping were properly executed. In fact, genetic and physical interactions have been described with the cap binding complex (CBC) components Cbp20 and Cbp80 (ref.…”

Influenza virus targets the mRNA export machinery and the nuclear pore complex

“…In addition, preferential interaction of mRNAs with certain RNA-binding proteins may dictate the fate of certain classes of mRNAs. In fact, it has been shown that different classes of mRNAs preferentially bind specific subsets of RNA-binding proteins (44), which could contribute to differential mRNA export. Thus, the selective nuclear retention of mRNAs encoding antiviral proteins in Nup98 and Rae1 mutant cells further indicate a role for these proteins in regulating innate and adaptive immunity and demonstrate the underlying complexity of the selective export of distinct mRNA species.…”

Recognition of polyadenosine RNA by zinc finger proteins

“…Although all conventional Pabs interact with RNA through RRM domains (24), there is evidence to suggest that at least one other type of RNA binding motif may confer specific binding to polyadenosine RNA (9,(25)(26)(27). The yeast protein, nuclear poly(A) binding protein 2 (Nab2), lacks RRM domains and instead contains two other potential RNA binding motifs, an arginine-glycineglycine (RGG) repeat domain and seven tandem CCCH zinc fingers (25,28).…”