Poster abstracts

Poster number 21 submitted by Melissa Hinman

Mammalian Hu protein family members have non-redundant functions as splicing suppressors

Melissa N. Hinman (Department of Genetics, Case Western Reserve University), Hui Zhu (Genomic Medicine Institute, Cleveland Clinic Foundation), Robert A. Hasman, Kavita Praveen (Department of Genetics, Case Western Reserve University), Hua Lou (Department of Genetics, Center for RNA Molecular Biology, Case Comprehensive Cancer Center, Case Western Reserve University)

Abstract:
Mammalian Hu proteins are a family of RNA-binding proteins that interact with AU-rich elements. Three of the four known family members, HuB, HuC, and HuD, are neuron-specific, while HuA (HuR in humans) is widely expressed. Hu proteins regulate mRNA stability and translation, and play an important role in neuronal differentiation. Our recent studies demonstrate a novel role for Hu proteins as splicing regulators that promote skipping of exon 23a of the neurofibromatosis type I (NF1) pre-mRNA.
Despite a high degree of sequence homology among the four members of the Hu family, it is predicted that they have distinct functional roles based on their expression patterns during development and in mature neurons. To determine if individual Hu members have differential effects on alternative splicing, we co-transfected cells with each member and a NF1 splicing reporter. Interestingly, individual Hu protein family members exhibit diverse potencies as splicing suppressors: HuR and mHuC strongly suppress NF1 exon 23a inclusion, hHuD has a moderate effect, and, most interestingly, mHuB has little effect. Immunofluorescence studies indicate that differential cellular localization is not responsible for the diverse splicing suppressor potencies of Hu family members.
To determine the amino acid basis of the differential effect of Hu proteins, we compared mHuB and mHuC sequences. Although the three RRM domains are highly homologous, the N-terminus and the hinge domain between RRM2 and RRM3 show great variability. When the N-terminal amino acids between the two proteins were swapped, the resulting fusion proteins acted like the parental proteins, indicating that the differential effect on splicing is not dictated by the sequence on the N-terminus of these proteins. We hypothesize that sequence variability in the hinge domain is responsible for the observed differential splicing suppressor strength. Our future experiments will focus on studying the role of the hinge domain in splicing regulation.

Keywords: Hu proteins, Alternative splicing, Neurofibromatosis type I