Garett M Padilla et al. (2004) Mid-west Worm Meeting "Investigating the Role of a Putative STAR Domain Protein in Caenorhabditis elegans"

Garett M Padilla, Elizabeth B Goodwin

[Mid-west Worm Meeting, 2004]

TRA-2 protein is necessary to promote female sexual development. Male development, at least in part, requires translational repression of tra-2 by two elements called TGEs ( tra-2 and GLI elements) located in the 3! (inverted exclamation mark)- (macron) UTR. tra-2 gain of function mutants that disrupt the TGEs feminize hermaphrodites. These mutations also feminize male animals, resulting in yolk production in the intestines and oocyte development in the germline. Therefore, TGE control regulates tra-2 activity in the germline and soma. GLD-1 is a germline specific protein that contains an RNA binding Maxi-KH/STAR domain that regulates tra-2 translation; however, a tra-2 somatic regulator has yet to be identified. We have identified a STAR domain containing protein in C. elegans , T21G5.5. This gene has two putative transcripts which differ in a 3! (inverted exclamation mark)- (macron) exon and is predicted to bind the same mRNAs as GLD-1 (Ryder et al , 2004). T21G5.5 are 63% identical throughout the STAR domain and 74% identical in the KH RNA binding domain. Both transcripts have been cloned and we are currently investigating their biochemical functions. Western analysis of a deletion strain of T21G5.5 demonstrates increased levels of TRA-2 protein relative to wildtype worms. In addition, RNA interference against T21G5.5a results in some in embryonic lethality and a decrease in fecundity. However, unlike GLD-1 gross germline phenotypes are not observed. Together these results indicate that T21G5.5 may function in the soma as a translational repressor in a role equivalent to GLD-1. Given slight germline defects observed, T21G5.5 may also function in the germline as well. Our future experiments include investigating the tissue specific and subcellular localization of T21G5.5, phenotypic analysis of the deletion strain, and determination of biochemical function. Ryder SP, Frater LA, Abramovitz DL, Goodwin EB, and Williamson JR. RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD- 1. Nature Structural and Molecular Biology 11 , 20-28 (2004).

Lehmann-Blount KA et al. (2005) J Mol Biol "Shape-specific nucleotide binding of single-stranded RNA by the GLD-1 STAR domain."

Williamson JR, Lehmann-Blount KA

[J Mol Biol, 2005]

Proteins containing the STAR RNA-binding domain fulfill vital roles in RNA biogenesis, yet a detailed understanding of STAR domain RNA binding specificity is lacking. In Caenorhabditis elegans, the STAR protein GLD-1 directly binds the 28 nucleotide recognition element TGE within the 3'' untranslated region of tra-2 mRNA. The GLD-1:TGE interaction promotes translational silencing of tra-2 mRNA, marking a pivotal event in the spermatogenesis to oogenesis switch in C.elegans hermaphrodites. By measuring the binding affinities of both GLD-1 and TGE mutants, we have explored the molecular determinants of STAR domain specificity. Site-directed GLD-1 mutants were guided by sequence homology with human splicing factor 1 (SF1), for which an RNA:protein complex structure is available in the work done by Liu et al. The RNA binding affinity of 11 mutant GLD-1 proteins was measured, and their binding specificity was assessed with a series of TGE RNAs containing natural or modified nucleotides. This combinatorial analysis of both RNA and protein mutants revealed a diverse array of specificities of individual nucleotide-binding pockets along the interface. At nucleotide position 18, adenosine appears to be specified by the overall shape of a pocket lined with aliphatic side-chains. At position 19, the high preference for cytidine is dependent on both the length of an amino acid side-chain and the identity of terminal functional groups. The nucleotide 21 binding pocket exhibits low discrimination for cytidine, and accommodates most nucleobases. The highly hydrophobic binding interface and apparent small number of hydrogen bonding read-out interactions at these positions is consistent with our finding that few amino acids seem to function individually in establishing binding specificity. Rather, specificity is conferred by the shape of the nucleotide-binding pocket. Our data provide the first detailed, quantitative analysis of the STAR domain, and highlight features of STAR:RNA recognition that are distinct among single-stranded RNA-binding proteins.

Ryder SP et al. (2008) Methods Mol Biol "Quantitative analysis of protein-RNA interactions by gel mobility shift."

Recht MI, Ryder SP, Williamson JR

[Methods Mol Biol, 2008]

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.

Recht MI et al. (2008) Methods Mol Biol "Monitoring assembly of ribonucleoprotein complexes by isothermal titration calorimetry."

Ryder SP, Williamson JR, Recht MI

[Methods Mol Biol, 2008]

Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studying RNA-protein interactions using ITC and gives specific examples for monitoring the binding of Caenorhabditis elegans GLD-1 STAR domain to TGE RNA and the binding of Aquifex aeolicus S6:S18 ribosomal protein heterodimer to an S15-ribosomal RNA complex.

Carmel AB et al. (2010) BMC Mol Biol "High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking."

Carmel AB, Lehmann-Blount KA, Williamson JR, Wu J

[BMC Mol Biol, 2010]

BACKGROUND: STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1. RESULTS: Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins. CONCLUSIONS: The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA binding by STAR proteins will help facilitate the identification of novel regulatory targets in vivo.

Wu J et al. (2013) J Mol Biol "A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex."

Williamson JR, Wu J, Campbell ZT, Menichelli E, Wickens M

[J Mol Biol, 2013]

The Pumilio and FBF (PUF) family of RNA-binding proteins interacts with protein partners to post-transcriptionally regulate mRNAs in eukaryotes. The interaction between PUF family member fem-3 binding factor (FBF) and germline development defective-3 (GLD-3) protein promotes spermatogenesis in Caenorhabditis elegans by increasing expression of the fem-3 mRNA. Defined here in these studies is the molecular basis for this critical interaction. A 10-amino-acid region within GLD-3 is required for FBF binding, while a 7-amino-acid loop in FBF between PUF repeats 7 and 8 is necessary for GLD-3 binding. These short sequences are conserved, as other FBF-binding proteins bear sequences similar to those in GLD-3 and other C. elegans PUF proteins contain sequences similar to those in FBF. The FBF-binding region of GLD-3 forms a ternary complex with FBF on the point mutation element (PME) in the fem-3 3' untranslated region, and formation of this GLD-3FBF complex does not impact the RNA-binding activity of FBF. These data raise the possibility of alternative models involving the formation of a GLD-3FBFRNA complex in the regulation of germline mRNAs.

Ryder SP et al. (2004) Nat Struct Mol Biol "RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1."

Goodwin EB, Ryder SP, Williamson JR, Abramovitz DL, Frater LA

[Nat Struct Mol Biol, 2004]

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.

Menichelli E et al. (2013) J Mol Biol "Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots."

Wu J, Menichelli E, Wickens M, Williamson JR, Campbell ZT

[J Mol Biol, 2013]

Caenorhabditis elegans CPB-1 (cytoplasmic polyadenylation element binding protein homolog-1) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms, CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a 10-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective-3). PUF proteins are characterized by the presence of eight -helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats 7 and 8. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.

Campbell ZT et al. (2012) J Biol Chem "Identification of a conserved interface between PUF and CPEB proteins."

Friend K, Menichelli E, Wu J, Campbell ZT, Williamson JR, Kimble J, Wickens M

[J Biol Chem, 2012]

Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.

Kerkow DE et al. (2012) J Mol Biol "The structure of the NXF2/NXT1 heterodimeric complex reveals the combined specificity and versatility of the NTF2-like fold."

Kerkow DE, Williamson JR, Menichelli E, Ambrus G, Hills RD, Gerace L, Carmel AB

[J Mol Biol, 2012]

NXF1-like members of the NXF (nuclear export factor) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate-specific and tissue-specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NXT (NTF2-related export) protein is central to NXF family function; however, given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remain unclear. Here, we report the structural and functional analyses of the Caenorhabditis elegans NXF variant ceNXF2 bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity, including a probable site for phosphoregulation, have been identified. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for the colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.