Identification of homologous protein models via 3D comparisons using predicted structures.

Recent advances in protein structure prediction enable 3D homology alignment and domain annotation using tertiary structures. Here, we present a protocol to identify homologous structures and annotate protein domains through in silico comparisons using the AlphaFold database. We describe steps for downloading and installing PyMOL software, preparing the query structure, and conducting a 3D homology search. The example provided highlights the application of this protocol in reevaluating an mpox viral protein annotation. For complete details on the use and execution of this protocol, please refer to Pan et al. (2023).1.

Unanticipated broad phylogeny of BEN DNA-binding domains revealed by structural homology searches.

Organization of protein sequences into domain families is a foundation for cataloging and investigating protein functions. However, long-standing strategies based on primary amino acid sequences are blind to the possibility that proteins with dissimilar sequences could have comparable tertiary structures. Building on our recent findings that in silico structural predictions of BEN family DNA-binding domains closely resemble their experimentally determined crystal structures, we exploited the AlphaFold2 database for comprehensive identification of BEN domains. Indeed, we identified numerous novel BEN domains, including members of new subfamilies. For example, while no BEN domain factors had previously been annotated in C. elegans, this species actually encodes multiple BEN proteins. These include key developmental timing genes of orphan domain status, sel-7 and lin-14, the latter being the central target of the founding miRNA lin-4. We also reveal that the domain of unknown function 4806 (DUF4806), which is widely distributed across metazoans, is structurally similar to BEN and comprises a new subtype. Surprisingly, we find that BEN domains resemble both metazoan and non-metazoan homeodomains in 3D conformation and preserve characteristic residues, indicating that despite their inability to be aligned by conventional methods, these DNA-binding modules are probably evolutionarily related. Finally, we broaden the application of structural homology searches by revealing novel human members of DUF3504, which exists on diverse proteins with presumed or known nuclear functions. Overall, our work strongly expands this recently identified family of transcription factors and illustrates the value of 3D structural predictions to annotate protein domains and interpret their functions.

On the exciton-assisted radiative recombination via impurity trap levels in AlGaN deep ultraviolet light-emitting diodes.

For decades, problems of parasitic emissions have been ubiquitously encountered in nearly all deep ultraviolet light-emitting diodes (DUV-LEDs). In this work, 450 nm parasitic peaks in 275 nm AlGaN DUV-LEDs have been studied in details. Upon careful comparisons and analyses on the electroluminescence and photoluminescence spectra at various injection levels and different temperatures, we have discovered a mechanism of exciton-assisted radiative recombination, namely, the reinforcement on radiative recombination via other impurity-trap levels (ITLs) by excitons that are generated in the midst of the band gap. For DUV-LED samples under investigation herein, a system of radiative ITLs within the band gap cannot be neglected. It includes two types of impurities located at two different energy levels, 3.80 eV and 2.75 eV, respectively. The former, establishing a sub-band edge, which behaves like an energy entrance to this system, contains a series of hydrogen-like excitons at a temperature lower than 100 K, which behaves like an energy entrance to this system. On the one hand, these excitons absorb carriers from band-edge and reduce the band-edge recombination. On the other hand they transfer the energy to lower impurity levels, enhancing the radiative recombination and giving rise to the 450 nm parasitic peak.

Multiple links between 5-methylcytosine content of mRNA and translation.

BACKGROUND: 5-Methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. RESULTS: We obtained ~ 1000 candidate m5C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m5C sites in mRNAs are enriched in 5'UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association. CONCLUSIONS: Our findings emphasise the major role of NSUN2 in placing the m5C mark transcriptome-wide. We further present evidence that substantiates a functional interdependence of cytosine methylation level with mRNA translation. Additionally, we identify several compelling candidate sites for future mechanistic analysis.

The 10-nm chromatin fiber and its relationship to interphase chromosome organization.

A chromosome is a single long DNA molecule assembled along its length with nucleosomes and proteins. During interphase, a mammalian chromosome exists as a highly organized supramolecular globule in the nucleus. Here, we discuss new insights into how genomic DNA is packaged and organized within interphase chromosomes. Our emphasis is on the structural principles that underlie chromosome organization, with a particular focus on the intrinsic contributions of the 10-nm chromatin fiber, but not the regular 30-nm fiber. We hypothesize that the hierarchical globular organization of an interphase chromosome is fundamentally established by the self-interacting properties of a 10-nm zig-zag array of nucleosomes, while histone post-translational modifications, histone variants, and chromatin-associated proteins serve to mold generic chromatin domains into specific structural and functional entities.