Published online 4 November 2008 Nucleic Acids Research, 2008, Vol. 36, No. 22 6977–6987 doi:10.1093/nar/gkn847 Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis Jan C. Scho¨ ning1, Corinna Streitner1, Irmtraud M. Meyer2,3, Yahong Gao1 and Dorothee Staiger1,* 1Molecular Cell Physiology, Bielefeld University, Bielefeld, Germany, 2UBC Bioinformatics Centre, Department of Computer Science and 3Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada Received June 25, 2008; Revised October 15, 2008; Accepted October 16, 2008 ABSTRACT The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post- transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5’ splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5’ half of the intron with a premature termination codon. In mutants defec- tive in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indi- cating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregu- latory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alter- native splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence con- servation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences. INTRODUCTION Post-transcriptional regulation has come into focus as an important mechanism to control gene expression in higher plants (1–4). It occurs at multiple levels including pre- mRNA maturation, mRNA transport, translation and breakdown. The major players are RNA-binding proteins that inﬂuence the fate of an mRNA molecule either directly by binding to deﬁned RNA sequences and struc- tural elements or indirectly through protein–protein inter- action (5). One important protein domain known to interact with RNA molecules is the RNA recognition motif (RRM) (6). It is composed of a four-stranded antiparallel b-sheet with two a-helices. The highly conserved octapeptide RNP1 and hexapeptide RNP2 sequence motifs are located in the b3 and b1 sheets and contain conserved aromatic resi- dues making contacts to the RNA substrate. A systematic survey disclosed 196 RRM-containing proteins in the genome of Arabidopsis thaliana (5). Among those, the 16 kDa AtGRP8 (A. thaliana glycine-rich RNA-binding pro- tein 8) protein combines a single N-terminal RRM with a C-terminal region enriched in glycine repeats with some interspersed serine, tyrosine and arginine residues (7–9). It is also known as GR-RBP8, GRP8 or CCR1 (cold and circadian regulated 1) (5,10,11). Both AtGRP8 and AtGRP7 encoding an orthologous RNA-binding protein that shares 77% sequence identity undergo circa- dian oscillations with a peak at the end of the daily light phase (9,11). Notably, AtGRP8 is subject to nega- tive regulation by AtGRP7, as in transgenic plants ectopically overexpressing AtGRP7 under control of the CaMV (Cauliﬂower Mosaic Virus) 35S RNA promoter, *To whom correspondence should be addressed. Tel: +49 521 106 5609; Fax: +49 521 106 6410; Email: dorothee.staiger@uni-bielefeld.de 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.