Heterogeneous nuclear ribonucleoprotein (hnRNP) H and F are members of a closely related subfamily of hnRNP proteins that are implicated in many aspects of RNA processing. hnRNP H and F are alternative splicing factors for numerous U2-and U12-dependent introns. The proteins have three RNA binding domains and two glycine-rich domains and localize to both the nucleus and cytoplasm, but little is known about which domains govern subcellular localization or splicing activity. We show here that the central glycine-tyrosine-arginine-rich (GYR) domain is responsible for nuclear localization, and a nonclassical nuclear localization signal (NLS) was mapped to a short, highly conserved sequence whose activity was compromised by point mutations. Glutathione S-transferase (GST) pulldown assays demonstrated that the hnRNP H NLS interacts with the import receptor transportin 1. Finally, we show that hnRNP H/F are transcription-dependent shuttling proteins. Collectively, the results suggest that hnRNP H and F are GYR domain-dependent shuttling proteins whose posttranslational modifications may alter nuclear localization and hence function.Most genes in metazoans contain multiple introns that need to be removed by mRNA splicing to generate mature mRNA. Alternative splicing, the process whereby multiple mRNAs (and thus proteins) are produced from the same gene through inclusion/exclusion of various exons (7), is widespread, and it has become clear that alternative splicing plays a large role in regulated gene expression and accounts for enormous protein diversity (9,38,62). The heterogeneous nuclear ribonucleoprotein (hnRNP) family of factors represents an important group of proteins that bind splicing regulatory elements to positively and negatively influence alternative splicing; hnRNP A1 and hnRNP H/F are particularly important in this regard (16-18, 42, 48, 50, 51). Binding to exonic splicing silencers and oligomerization of hnRNP A1 along the exon are thought to sterically displace positively acting factors, such as serine-arginine (SR) proteins (61, 82), or alternatively, hnRNP A1 may operate to repress splicing by binding to intronic splicing silencers, which may loop out the exon (8, 60). This looping mechanism might also account for a positive role for hnRNP A1 and H/F in splicing control through interactions with sites at each end of long introns, thus bringing splice sites together (24,47).In addition to their influence on splicing through recognition of specific binding sites within target RNAs, subcellular localization of hnRNP proteins has emerged as a mechanism to regulate splicing. While essentially nuclear at steady state, several hnRNP proteins (hnRNP A1/A2, K, D, E, and I) continuously shuttle between the nucleus and cytoplasm (55). hnRNP A1 is particularly well studied in this regard and shuttles through the action of a bidirectional transport peptide called M9 (65). While the export activity of M9 is not well understood, nuclear import of hnRNP A1 is mediated by the interaction of the M9 peptide with the imp...