The gel mobility shift assay is routinely used to visualize protein-RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. We review the quantitative application of this approach to elucidate thermodynamic properties of protein-RNA complexes. Assay designs for titration, competition, and stoichiometry experiments are presented for two unrelated model complexes.

RNA-binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP-mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD-1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD-1 interactions with the transcriptome, we identified GLD-1-associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD-1 association with mRNA is determined by the strength and number of 7-mer GLD-1-binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD-1/GBM-binding experiments to determine relative affinity, and in vivo, by 'transplantation' experiments, where 'weak' and 'strong' GBMs imposed translational repression of increasing strength on a non-target mRNA. This study demonstrates that transcriptome-wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post-transcriptional regulatory networks.

The post-transcriptional regulation of gene expression underlies several critical developmental phenomena. In metazoa, gene products that are expressed, silenced and packaged during oogenesis govern early developmental processes prior to nascent transcription activation. Furthermore, tissue-specific alternative splicing of several transcription factors controls pattern formation and organ development. A highly conserved family of proteins containing a STAR/GSG RNA-binding domain is essential to both processes. Here, we identify the consensus STAR-binding element (SBE) required for specific mRNA recognition by GLD-1, a key regulator of Caenorhabditis elegans germline development. We have identified and verified new GLD-1 repression targets containing this sequence. The results suggest additional functions of GLD-1 in X-chromosome silencing and early embryogenesis. The SBE is present in Quaking and How mRNA targets, suggesting that STAR protein specificity is highly conserved. Similarities between the SBE and the branch-site signal indicate a possible competition mechanism for STAR/GSG regulation of splicing variants.

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9 -13-nucleotide window. This sequence is remarkably abundant in the 3-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein.We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation. Embryogenesis is the process by which a fertilized oocyte transforms into a multicellular organism. Although the zygote contains all of the information required for development, zygotic DNA alone is not sufficient to drive patterning. Somatic cell nuclear transfer experiments, like those used to clone Dolly the sheep, demonstrate that maternal factors present in the oocyte cytoplasm are needed for the initiation of development (1). These maternal factors are proteins and quiescent mRNAs (2); they coordinate early development prior to the onset of zygotic transcription.In the nematode worm Caenorhabiditis elegans, polarization of the body axes occurs after fertilization and requires several highly conserved maternal factors termed PAR proteins (3-10). Prior to fertilization, these proteins are uniformly distributed in the cytoplasm. Once the sperm penetrates the oocyte, they localize to opposing cortical domains in a process that requires microtubules derived from the asters of the sperm pronucleus. The PAR network coordinates asymmetric translation of several cell signaling proteins (11, 12) (glp-1, apx-1, mom-2, and mom-5) and transcription factors (13) (skn-1, pal-1, and pop-

Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homo-dimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may impact function in the APOBEC3 enzymes, and provides a potential scaffold for strategies aimed at reducing their mutation load.