SignificanceWe present identification of the luciferase and enzymes of the biosynthesis of a eukaryotic luciferin from fungi. Fungi possess a simple bioluminescent system, with luciferin being only two enzymatic steps from well-known metabolic pathways. The expression of genes from the fungal bioluminescent pathway is not toxic to eukaryotic cells, and the luciferase can be easily co-opted to bioimaging applications. With the fungal system being a genetically encodable bioluminescent system from eukaryotes, it is now possible to create artificially bioluminescent eukaryotes by expression of three genes. The fungal bioluminescent system represents an example of molecular evolution of a complex ecological trait and with molecular details reported in the paper, will allow additional research into ecological significance of fungal bioluminescence.

Autoluminescent plants that express a bacterial bioluminescence gene cluster 1 have not been widely adopted due to requisite expression in plastids and low light output. Alternatively, we have engineered tobacco lines expressing a fungal bioluminescent system 2 , which converts caffeic acid present in all plants into luciferin, and report self-sustained luminescence easily visible to the naked eye. Our findings might underpin development of a suite of imaging tools for plants.

Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.

Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

In contrast to fluorescent proteins, light emission from luciferase reporters requires exogenous addition of a luciferin substrate. Bacterial bioluminescence has been the single exception, where an operon of five genes is sufficient to produce light autonomously. Although commonly used in prokaryotic hosts, toxicity of the aldehyde substrate has limited its use in eukaryotes 1 . Here we demonstrate autonomous luminescence in a multicellular eukaryotic organism by incorporating a recently discovered fungal bioluminescent system 2 into tobacco plants. We monitored these light-emitting plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. The dynamic patterns of luminescence reflected progression through developmental stages, circadian oscillations, transport, and response to injuries. As with other fluorescent and luminescent reporters, we anticipate that this system will be further engineered for varied purposes, especially where exogenous addition of substrate is undesirable.

Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L – a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins – silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed.