Massively parallel identification of mRNA localization elements in primary cortical neurons.

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called 'zipcodes'. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3' untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons-cell bodies and neurites-with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes.

Nuclear export determines the cytokine sensitivity of STAT transcription factors.

Cytokine-dependent gene activation critically depends upon the tyrosine phosphorylation (activation) of STAT transcription factors at membrane-bound cytokine receptors. The extent of STAT activation and hence the specificity of signaling is primarily determined by structural complementarity between the SH2 domain of the STATs and the tyrosine-phosphorylated receptor chains. Here, we identified constitutive nucleocytoplasmic shuttling as another mechanism that controls the differential activation of STAT transcription factors. Our analysis of nucleocytoplasmic cycling of STAT1 revealed that the expression of the alternatively spliced transactivation domain and its signal-dependent serine phosphorylation maximized the rate of nuclear export. Export modulation occurred independently of retention factors or the export receptor CRM1, and was observed both before and during stimulation of cells with cytokines. Our data indicated a dual role for the transactivation domain. It enhanced the nuclear retention of activated STAT1, but had the opposite effect on inactivated molecules. Accordingly, and despite their identical receptor recognition, the STAT1 splice variants differed strongly in the amplitude of tyrosine phosphorylation and in the duration of the cytokine signal. Thus, regulated nuclear export determined the cytokine sensitivity of the shuttling STAT1 transcription factors by controlling their availability at the receptor kinase complex.

Constitutive and IFN-gamma-induced nuclear import of STAT1 proceed through independent pathways.

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. Here, we identify and characterize a non-transferable nuclear targeting sequence in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-gamma (IFN-gamma)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-gamma stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss-of-function mutations of the dimer-specific import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-alpha-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.