Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells.

A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex.

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.

Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots.

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-1⋅FBF complex are not known.

Identification of a conserved interface between PUF and CPEB proteins.

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.

High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking.

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.

Chloroplast translation regulation.

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell.

Chlamydomonas reinhardtii chloroplasts as protein factories.

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce.

Control of translocations between highly diverged genes by Sgs1, the Saccharomyces cerevisiae homolog of the Bloom's syndrome protein.

Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3.

Inhibition of NF-kappaB activity by IkappaBbeta in association with kappaB-Ras.

IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein.

KappaB-Ras binds to the unique insert within the ankyrin repeat domain of IkappaBbeta and regulates cytoplasmic retention of IkappaBbeta x NF-kappaB complexes.

The IkappaBalpha and IkappaBbeta proteins inhibit the transcriptional potential of active NF-kappaB dimers through stable complex formation. It has been shown that inactive IkappaBalpha x NF-kappaB complexes shuttle in and out of the nucleus, whereas IkappaBbeta x NF-kappaB complexes are retained exclusively in the cytoplasm of resting cells. The biochemical mechanism underlying this functional difference and its consequences are unknown.