An integral step of gene expression is the formation of an mRNP by the assembly of nuclear RNA-binding proteins (RBPs) onto the mRNA. This mRNP assembly is necessary for mRNA stability, and only correctly packaged mRNPs are exported from the nucleus through the nuclear pore complexes to the cytoplasm. In addition, bound RBPs often regulate cytoplasmic processes, such as mRNA localization, translation and decay. Thus, the formation and composition of an mRNP is important for the posttranscriptional control of gene expression.
The functions of the RBPs involved in mRNP assembly have been largely analyzed by deletion or depletion of the whole protein or at least protein domains, which probably abrogates several functions of each protein at once. In order to determine specifically the RNA-binding function of proteins involved in nuclear mRNP assembly, we first determined the amino acids binding to RNA in vivo by cross-linking and mass spectrometry (RNPXL). We identified about 100 amino acids cross-linked to RNA in vivo in components of the nuclear mRNP. Second, we can now specifically elucidate the function of the RNA-binding activity of these proteins by mutation of the identified amino acids. Here, we present the analysis of RNA-binding sites in the protein Npl3, an SR-like protein with functions in transcription elongation, splicing, 3' end formation, mRNP assembly, and nuclear mRNA export. Interestingly, abrogation of mRNA-binding in different regions of Npl3 leads to different phenotypes.
Taken together, we identify the in vivo RNA binding sites of nuclear mRNA binding proteins involved in mRNP assembly and nuclear mRNA export. In addition, we show that abrogation of RNA-binding in different domains of the protein Npl3 has specific and surprisingly different functional consequences. Thus, our approach unravels novel and unexpected insights into the process of nuclear mRNP assembly.