Gemin5 delivers snRNA precursors to the SMN complex for snRNP biogenesis.

The SMN complex assembles Sm cores on snRNAs, a key step in the biogenesis of snRNPs, the spliceosome's major components. Here, using SMN complex inhibitors identified by high-throughput screening and a ribo-proteomic strategy on formaldehyde crosslinked RNPs, we dissected this pathway in cells. We show that protein synthesis inhibition impairs the SMN complex, revealing discrete SMN and Gemin subunits and accumulating an snRNA precursor (pre-snRNA)-Gemin5 intermediate. By high-throughput sequencing of this transient intermediate's RNAs, we discovered the previously undetectable precursors of all the snRNAs and identified their Gemin5-binding sites. We demonstrate that pre-snRNA 3' sequences function to enhance snRNP biogenesis. The SMN complex is also inhibited by oxidation, and we show that it stalls an inventory-complete SMN complex containing pre-snRNAs. We propose a stepwise pathway of SMN complex formation and snRNP biogenesis, highlighting Gemin5's function in delivering pre-snRNAs as substrates for Sm core assembly and processing.

RNA-binding proteins and post-transcriptional gene regulation.

RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization. Eukaryotic cells encode a large number of RBPs (thousands in vertebrates), each of which has unique RNA-binding activity and protein-protein interaction characteristics. The remarkable diversity of RBPs, which appears to have increased during evolution in parallel to the increase in the number of introns, allows eukaryotic cells to utilize them in an enormous array of combinations giving rise to a unique RNP for each RNA. In this short review, we focus on the RBPs that interact with pre-mRNAs and mRNAs and discuss their roles in the regulation of post-transcriptional gene expression.

Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains.

Gemin5 binds specifically to the small nuclear RNA (snRNA)-defining small nuclear ribonucleoprotein (snRNP) code sequence and is essential, together with other components of the survival of motor neurons (SMN) complex, for the biogenesis of snRNPs, the major constituents of spliceosomes. We show that this binding is mediated by Gemin5's WD repeat domain, a common domain not previously known to bind RNA independently. The entire WD repeat domain, comprising 13 WD motifs, is both necessary and sufficient for sequence-specific, high-affinity binding of Gemin5 to its RNA targets. Using an RNA-mediated hydroxyl radical probing method and mass spectrometry, we mapped a discrete region of the WD repeat domain that contacts snRNAs and demonstrated by mutagenesis that specific amino acids in this region are crucial for Gemin5-snRNA binding. The WD repeat domain is thus a previously undescribed RNA binding domain, and we suggest that the presence of WD repeats should be considered as predictive of potential function in RNA binding.

Antagonism between GLD-2 binding partners controls gamete sex.

Cytoplasmic polyadenylation is a key mechanism of gene control. In Caenorhabditis elegans, GLD-2 and GLD-3 provide the catalytic and RNA-binding subunits, respectively, of a major cytoplasmic poly(A) polymerase (PAP). Here, we identify RNP-8 as a second GLD-2 partner. RNP-8 binds GLD-2 and stimulates GLD-2 activity to form a functional PAP, much like GLD-3. Moreover, GLD-2/RNP-8 and GLD-2/GLD-3 exist as separate complexes that form selectively during development, and RNP-8 and GLD-3 appear to have distinct RNA-binding specificities. Therefore, GLD-2 can form either of two discrete PAPs. In C. elegans hermaphrodites, gamete production begins with spermatogenesis and transitions later to oogenesis. We suggest that the combinatorial use of GLD-2 contributes to this transition, as GLD-2/GLD-3 promotes spermatogenesis, whereas GLD-2/RNP-8 specifies oogenesis. Indeed, RNP-8 and GLD-3 antagonize each other, as evidenced by genetic cosuppression and molecular competition for GLD-2 binding. We conclude that GLD-2 and its binding partners control gamete identity.

Redundant control of the Caenorhabditis elegans sperm/oocyte switch by PUF-8 and FBF-1, two distinct PUF RNA-binding proteins.

PUF proteins control both growth and differentiation in the C. elegans germ line. These conserved RNA-binding proteins inhibit expression of target mRNAs, either by repressing translation or promoting degradation. Previous studies showed that PUF-8, a PUF protein with striking similarity to human Pumilio, prevents return of primary spermatocytes to the mitotic cell cycle [Subramaniam, K. & Seydoux, G. (2003) Curr. Biol. 13, 134-139]. We now report that PUF-8 is also critical for the hermaphrodite sperm/oocyte switch. Most puf-8 mutant hermaphrodites make both sperm and oocytes and are self-fertile, but some make a vast excess of sperm and fail to switch into oogenesis. This puf-8 defect is dramatically enhanced by removal of another puf gene called fbf-1: all fbf-1 puf-8 double mutants fail in the hermaphrodite sperm/oocyte switch. Therefore, puf-8 and fbf-1 act redundantly to control this decision. Epistasis analyses place puf-8 and fbf-1 upstream of fog-2, a gene near the top of the germ-line sex determination pathway. Furthermore, the abundance of FOG-2 increases dramatically in the distal region of fbf-1 puf-8 double mutants. We suggest that PUF-8 and FBF-1 may control fog-2 expression, and that the sperm/oocyte decision occurs in the distal germ line.

Dose-dependent control of proliferation and sperm specification by FOG-1/CPEB.

RNA-binding proteins control germline development in metazoans. This work focuses on control of the C. elegans germline by two RNA-binding proteins: FOG-1, a CPEB homolog; and FBF, a PUF family member. Previous studies have shown that FOG-1 specifies the sperm fate and that FBF promotes proliferation. Here, we report that FOG-1 also promotes proliferation. Whereas fbf-1 fbf-2 double mutants make approximately 120 germ cells, fog-1; fbf-1 fbf-2 triple mutants make only approximately 10 germ cells. The triple mutant germline divides normally until early L2, when germ cells prematurely enter meiosis and begin oogenesis. Importantly, fog-1/+; fbf-1 fbf-2 animals make more germ cells than fbf-1 fbf-2 double mutants, demonstrating that one dose of wild-type fog-1 promotes proliferation more effectively than two doses - at least in the absence of FBF. FOG-1 protein is barely detectable in proliferating germ cells, but abundant in germ cells destined for spermatogenesis. Based on fog-1 dose effects, together with the gradient of FOG-1 protein abundance, we suggest that low FOG-1 promotes proliferation and high FOG-1 specifies spermatogenesis. FBF binds specifically to regulatory elements in the fog-1 3'UTR, and FOG-1 increases in animals lacking FBF. Therefore, FBF represses fog-1 expression. We suggest that FBF promotes continued proliferation, at least in part, by maintaining FOG-1 at a low level appropriate for proliferation. The dose-dependent control of proliferation and cell fate by FOG-1 has striking parallels with Xenopus CPEB, suggesting a conserved mechanism in animal development.

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans.

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.