Background: ELAV-like neuronal RNA-binding proteins are highly conserved in many neuronecontaining organisms and have been implicated in neuronal development and differentiation.

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.

Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2␤ (hTra2␤; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2␤, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model ␤-globin pre-mRNA and a human tra-2␤ pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase ␥-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.Most pre-mRNAs in higher eukaryotes complete accurate splicing in the nucleus, as a prerequisite for carrying the correct genetic information to the cytoplasm for translation. Constitutive splicing is highly precise and is sensitive to mutations in critical signal sequences of the pre-mRNA. Indeed, human genetic diseases are often caused by point mutations that lie in 5Ј or 3Ј splice site elements or by creation of new ones at inappropriate locations, which result in splicing defects (reviewed in references 33 and 50). On the other hand, a subset of pre-mRNAs shows sufficient flexibility for alternative potential splice sites to be used, often in a regulated way in response to tissue-specific or developmentally regulated states (reviewed in references 24, 55, and 82). This process, called alternative splicing, is a basic strategy for the regulation of eukaryotic gene expression. An unexpectedly small set of protein-coding genes, at most 30,000 has recently been estimated in the human genome (51, 72, 79). Indeed, a higher prevalence of alternative splicing than earlier estimates is likely responsible for a larger number of, and more complex, protein products (51, 64).Pre-mRNA splicing takes place within a large complex, or spliceosome, which includes the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4/U6, and U5, together with a large number of non-snRNP protein factors. Biochemical characterization of the spliceosome, together with genetic studies in fission and budding yeast, predicted that more than 50 proteins are essential for constitutive splicing (reviewed in references 15 and 73). The members of the serine/arginine-rich protein (SR protein) family are wel...

Hu proteins are RNA-binding proteins that are the vertebrate homologs of Drosophila ELAV, and are implicated in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Here, using the yeast two-hybrid system, we show that neuron-specific Hu proteins can interact with themselves. Immuno precipitation assays demonstrated that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA. Furthermore, using in situ chemical crosslinking assays, we found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs.