No abstract

SummaryThe clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein in¯uences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5 H cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein speci®cally interacts with the 3 H untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also in¯uences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

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Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)؉ RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3 untranslated region (3-UTR) and protecting the mRNA from exonucleolytic degradation . To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3-UTRs and contributing to the stabilization of mRNAs in the nucleus.Newly synthesized pre-mRNAs associate with a set of proteins known as heterogeneous nuclear RNA-binding proteins (hnRNPs). In human cells, more than 20 abundant hnRNPs, designated A1 through U, have been identified (reviewed in references 15, 33, 57, and 61). Similar proteins are also present in other vertebrates. In Drosophila melanogaster, 12 major proteins, designated hrp36 through hrp75, have been identified (26,27,28,40,42). They share considerable sequence similarity with human hnRNPs (26,27,28,40,42). Some hnRNP-like proteins have been identified in Saccharomyces cerevisiae (1,10,32,54,64).Originally, hnRNPs were believed to be responsible only for packaging and proper folding of processing substrates (reviewed in reference 57). However, more recently, many of the hnRNPs have been identified as factors involved in different stages of mRNA maturation (reviewed in references 33, 56, and 61). They have been shown to play roles in pre-mRNA splicing; 3Ј-end formation; mRNA transport, translation, and stability; and transcription regulation (reviewed in reference 33). Notably, the same hnRNP can influence mRNA maturation at different levels. For example, hnRNP A1 is involved in alternative splicing (8,12,44) and mRNA export (30, 45, 60; reviewed in reference 47). It is generally accepted that hnRNPs bind to the nascent RNA in a transcript-specific way, resulting in a unique comb...