Sean P. Ryder

RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1

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.

Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site

Helix packing is critical for RNA tertiary structure formation, although the rules for helix–helix association within structured RNAs are largely unknown. Docking of the substrate helix into the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report that complementary sets of noncanonical base pairs (a G·U wobble pair and two consecutively stacked sheared A·A pairs) create an RNA helix packing motif that is essential for 5′-splice site selection in the group I intron. This is likely to be a general motif for helix–helix interaction within the tertiary structures of many large RNAs.

Quantitative analysis of protein-RNA interactions by gel mobility shift.

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.

Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

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.

A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1.

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.

Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes.

Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods.

[6] Chemical probing of RNA by nucleotide analog interference mapping

Publisher Summary This chapter outlines the synthesis of phosphorothioate-tagged analogs and their in vitro incorporation into RNA transcripts. It also describes an interference mapping assay using a reverse splicing form of the tetrahymena group I ribozyme. This includes a procedure for the quantitation of the interference data and an interpretation of characteristic interference patterns. Nucleotide analog interference mapping (NAIM) is a chemogenetic approach that rapidly identifies functional groups that are important for the activity of RNA. NAIM utilizes a pool of randomly substituted RNAs generated by in vitro transcription, and the active RNAs are identified through a selection experiment. In this way, a particular RNA can be screened with multiple analogs in a time efficient manner. NAIM is applicable to any RNA with a known function. It can be used to study RNA catalysis through cleavage or ligation, RNA interactions with protein and other ligands, and the steps of RNA folding. This approach should make it possible to identify the chemical groups important for a wide variety of RNA and potentially DNA activities.

The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p.

The Sec1/Munc18 (SM) protein family regulates intracellular trafficking through interactions with individual SNARE proteins and assembled SNARE complexes. Revealing a common mechanism of this regulation has been challenging, largely because of the multiple modes of interaction observed between SM proteins and their cognate syntaxin-type SNAREs. These modes include binding of the SM to a closed conformation of syntaxin, binding to the N-terminal peptide of syntaxin, binding to assembled SNARE complexes, and/or binding to nonsyntaxin SNAREs. The SM protein Vps45p, which regulates endosomal trafficking in yeast, binds the conserved N-terminal peptide of the syntaxin Tlg2p. We used size exclusion chromatography and a quantitative fluorescent gel mobility shift assay to reveal an additional binding site that does not require the Tlg2p N-peptide. Characterization of Tlg2p mutants and truncations indicate that this binding site corresponds to a closed conformation of Tlg2p. Furthermore, the Tlg2p N-peptide competes with the closed conformation for binding, suggesting a fundamental regulatory mechanism for SM–syntaxin interactions in SNARE assembly and membrane fusion.

Quaking Regulates Hnrnpa1 Expression through Its 3′ UTR in Oligodendrocyte Precursor Cells

In mice, Quaking (Qk) is required for myelin formation; in humans, it has been associated with psychiatric disease. QK regulates the stability, subcellular localization, and alternative splicing of several myelin-related transcripts, yet little is known about how QK governs these activities. Here, we show that QK enhances Hnrnpa1 mRNA stability by binding a conserved 3′ UTR sequence with high affinity and specificity. A single nucleotide mutation in the binding site eliminates QK-dependent regulation, as does reduction of QK by RNAi. Analysis of exon expression across the transcriptome reveals that QK and hnRNP A1 regulate an overlapping subset of transcripts. Thus, a simple interpretation is that QK regulates a large set of oligodendrocyte precursor genes indirectly by increasing the intracellular concentration of hnRNP A1. Together, the data show that hnRNP A1 is an important QK target that contributes to its control of myelin gene expression.

RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3

Totipotent stem cells have the potential to differentiate into every cell type. Renewal of totipotent stem cells in the germline and cellular differentiation during early embryogenesis rely upon posttranscriptional regulatory mechanisms. The Caenorhabditis elegans RNA binding protein, MEX-3, plays a key role in both processes. MEX-3 is a maternally-supplied factor that controls the RNA metabolism of transcripts encoding critical cell fate determinants. However, the nucleotide sequence specificity and requirements of MEX-3 mRNA recognition remain unclear. Only a few candidate regulatory targets have been identified, and the full extent of the network of MEX-3 targets is not known. Here, we define the consensus sequence required for MEX-3 RNA recognition and demonstrate that this element is required for MEX-3 dependent regulation of gene expression in live worms. Based on this work, we identify several candidate MEX-3 targets that help explain its dual role in regulating germline stem cell totipotency and embryonic cell fate specification.