Brian M. Farley

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.

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.

Regulation of maternal mRNAs in early development.

Most sexually reproducing metazoans are anisogamous, meaning that the two gametes that combine during fertilization differ greatly in size. By convention, the larger gametes are considered female and are called ova, while the smaller gametes are male and are called sperm. In most cases, both gametes contribute similarly to the chromosomal content of the new organism. In contrast, the maternal gamete contributes nearly all of the cytoplasm. This cytoplasmic contribution is crucial to patterning early development; it contains the maternal proteins and transcripts that guide the early steps of development prior to the activation of zygotic transcription. This review compares and contrasts early development in common laboratory model organisms in order to highlight the similarities and differences in the regulation of maternal factors. We will focus on the production and reversible silencing of maternal mRNAs during oogenesis, their asymmetric activation after fertilization, and their subsequent clearance at the midblastula transition. Where possible, insights from mechanistic studies are presented.

FBF represses the Cip/Kip cell‐cycle inhibitor CKI‐2 to promote self‐renewal of germline stem cells in C. elegans

Although the decision between stem cell self‐renewal and differentiation has been linked to cell‐cycle modifications, our understanding of cell‐cycle regulation in stem cells is very limited. Here, we report that FBF/Pumilio, a conserved RNA‐binding protein, promotes self‐renewal of germline stem cells by repressing CKI‐2Cip/Kip, a Cyclin E/Cdk2 inhibitor. We have previously shown that repression of CYE‐1 (Cyclin E) by another RNA‐binding protein, GLD‐1/Quaking, promotes germ cell differentiation. Together, these findings suggest that a post‐transcriptional regulatory circuit involving FBF and GLD‐1 controls the self‐renewal versus differentiation decision in the germline by promoting high CYE‐1/CDK‐2 activity in stem cells, and inhibiting CYE‐1/CDK‐2 activity in differentiating cells.

Post-transcriptional regulation of myelin formation

Myelin is a specialized structure of the nervous system that both enhances electrical conductance and protects neurons from degeneration. In the central nervous system, extensively polarized oligodendrocytes form myelin by wrapping cellular processes in a spiral pattern around neuronal axons. Myelin formation requires the oligodendrocyte to regulate gene expression in response to changes in its extracellular environment. Because these changes occur at a distance from the cell body, post-transcriptional control of gene expression allows the cell to fine-tune its response. Here, we review the RNA-binding proteins that control myelin formation in the brain, highlighting the molecular mechanisms by which they control gene expression and drawing parallels from studies in other cell types.

Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci

Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using Library MosSCI

Background: In C. elegans, germline development and early embryogenesis rely on posttranscriptional regulation of maternally transcribed mRNAs. In many cases, the 3′ untranslated region (UTR) is sufficient to govern the expression patterns of these transcripts. Several RNA‐binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA‐binding protein–mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA‐binding proteins and their associated mRNAs. Results: In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RNA‐binding proteins that control the expression pattern of five different maternal mRNAs. Conclusions: The results provide a generalizable and efficient strategy to assess the functional relevance of protein–RNA interactions in vivo, and reveal new regulatory connections between key RNA‐binding proteins and their maternal mRNA targets. Developmental Dynamics 245:925–936, 2016.