Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein-mRNA tethering assay to probe the function of an RNA binding ...protein in its natural context within the
adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The
reporter 3'UTR harbors functional binding elements for λN22 peptide, while the
reporter 3'UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a
Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its amino-terminal intrinsically disordered region, not its carboxy-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: Classically Nanos proteins are expected to repress RNA, but
had been found to promote
expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RNA binding protein tethering to determine its molecular mechanism in a multicellular tissue.
Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle ...reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 Å) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal "arms" of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca²⁺-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.
Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca²⁺) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and ...initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca²⁺ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.
Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. ...Using nematode “P-granules” as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, singlestranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds.
The superior carrier mobility of SiGe alloys make them a highly desirable channel material in complementary metal-oxide-semiconductor (CMOS) transistors. Passivation of the SiGe surface and the ...associated minimization of interface defects between SiGe channels and high-k dielectrics continues to be a challenge for fabrication of high-performance SiGe CMOS. A primary source of interface defects is interfacial GeO x . This interfacial oxide can be decomposed using an oxygen-scavenging reactive gate metal, which nearly eliminates the interfacial oxides, thereby decreasing the amount of GeO x at the interface; the remaining ultrathin interlayer is consistent with a SiO x -rich interface. Density functional theory simulations demonstrate that a sub-0.5 nm thick SiO x -rich surface layer can produce an electrically passivated HfO2/SiGe interface. To form this SiO x -rich interlayer, metal gate stack designs including Al/HfO2/SiGe and Pd/Ti/TiN/nanolaminate (NL)/SiGe (NL: HfO2–Al2O3) were investigated. As compared to the control Ni-gated devices, those with Al/HfO2/SiGe gate stacks demonstrated more than an order of magnitude reduction in interface defect density with a sub-0.5 nm SiO x -rich interfacial layer. To further increase the oxide capacitance, the devices were fabricated with a Ti oxygen scavenging layer separated from the HfO2 by a conductive TiN diffusion barrier (remote scavenging). The Pd/Ti/TiN/NL/SiGe structures exhibited significant capacitance enhancement along with a reduction in interface defect density.
FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we ...determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3′ UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression.
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•FOG-3 crystal structure reveals sites of missense mutations•FOG-3 assembles into dimers that can multimerize•FOG-3 binds directly to mRNAs in the oogenic program•FOG-3 recruited to a reporter mRNA represses its expression
The mechanism of the sperm or oocyte fate decision has been elusive. Aoki et al. report that nematode FOG-3, a Tob/BTG protein driving sperm fate, has evolved from monomeric to multimeric form with acquisition of a divergent Tob/BTG mechanism for mRNA repression.
Germ cell specification as sperm or oocyte is an ancient cell fate decision, but its molecular regulation is poorly understood. In Caenorhabditis elegans, the FOG-1 and FOG-3 proteins behave ...genetically as terminal regulators of sperm fate specification. Both are homologous to well-established RNA regulators, suggesting that FOG-1 and FOG-3 specify the sperm fate post-transcriptionally. We predicted that FOG-1 and FOG-3, as terminal regulators of the sperm fate, might regulate a battery of gamete-specific differentiation genes. Here we test that prediction by exploring on a genomic scale the messenger RNAs (mRNAs) associated with FOG-1 and FOG-3. Immunoprecipitation of the proteins and their associated mRNAs from spermatogenic germlines identifies 81 FOG-1 and 722 FOG-3 putative targets. Importantly, almost all FOG-1 targets are also FOG-3 targets, and these common targets are strongly biased for oogenic mRNAs. The discovery of common target mRNAs suggested that FOG-1 and FOG-3 work together. Consistent with that idea, we find that FOG-1 and FOG-3 proteins co-immunoprecipitate from both intact nematodes and mammalian tissue culture cells and that they colocalize in germ cells. Taking our results together, we propose a model in which FOG-1 and FOG-3 work in a complex to repress oogenic transcripts and thereby promote the sperm fate.
The superior carrier mobility of SiGe alloys make them a highly desirable channel material in complementary metal-oxide-semiconductor (CMOS) transistors. Passivation of the SiGe surface and the ...associated minimization of interface defects between SiGe channels and high- k dielectrics continues to be a challenge for fabrication of high-performance SiGe CMOS. A primary source of interface defects is interfacial GeO
. This interfacial oxide can be decomposed using an oxygen-scavenging reactive gate metal, which nearly eliminates the interfacial oxides, thereby decreasing the amount of GeO
at the interface; the remaining ultrathin interlayer is consistent with a SiO
-rich interface. Density functional theory simulations demonstrate that a sub-0.5 nm thick SiO
-rich surface layer can produce an electrically passivated HfO
/SiGe interface. To form this SiO
-rich interlayer, metal gate stack designs including Al/HfO
/SiGe and Pd/Ti/TiN/nanolaminate (NL)/SiGe (NL: HfO
-Al
O
) were investigated. As compared to the control Ni-gated devices, those with Al/HfO
/SiGe gate stacks demonstrated more than an order of magnitude reduction in interface defect density with a sub-0.5 nm SiO
-rich interfacial layer. To further increase the oxide capacitance, the devices were fabricated with a Ti oxygen scavenging layer separated from the HfO
by a conductive TiN diffusion barrier (remote scavenging). The Pd/Ti/TiN/NL/SiGe structures exhibited significant capacitance enhancement along with a reduction in interface defect density.
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