Biology subjects

Jayachandran, U.

Publications and source records attributed to Jayachandran, U..

5 recordsLinked to original sources

bioRxiv2026

The fungal RNA-binding protein Ssd1 represses Sun4 protein abundance through recognition of 5' UTR structural and sequence elements

Regulation of protein abundance allows fungi to adapt to changing environments, regulate their growth and morphology, and react to external stresses. Ssd1 is a fungal RNA binding protein that binds mRNAs encoding cell wall remodelling proteins and regulates cell wall biogenesis. Ssd1 binding sites (SBSs) have been described by computational and biochemical analyses, but how Ssd1 recognises these sites and how that relates to regulation of protein abundance was less clear. Here, a co-crystal structure of Saccharomyces cerevisiae Ssd1 with an SBS reveals core determinants of recognition, while fluorescent reporters of Sun4, an Ssd1-regulated cell wall protein, were used to characterise structure- guided mutations. We find that Ssd1 has an extensive RNA binding site that recognises two elements of the SBS: an upstream element that forms a structural motif, and an element containing tandem CNYU sequences that engages Ssd1 in base-specific recognition. Mutations to the Ssd1 RNA binding surface prevent Ssd1-dependent repression of fluorescent reporters and show strong phenotypes in assays of cell wall stress resistance and genetic interactions with the Cbk1 kinase. Loss of repression is also observed if both SUN4 SBSs are altered. However, the presence of one SBS in the SUN4 5' untranslated region is sufficient to confer Ssd1-dependent suppression of protein abundance. Our work confirms that RNA binding is a core function of Ssd1 and is likely to inform functional analyses in fungi beyond S. cerevisiae, where Ssd1 orthologs have been identified as virulence factors in several fungal pathogens.

molecular biology↗

bioRxiv2024

Integrative structural analysis of NF45-NF90 heterodimers reveals architectural rearrangements and oligomerisation on binding dsRNA.

Complexes of nuclear factors 45 and 90 (NF45-NF90) play a multitude of roles in co- and post-transcriptional RNA processing, including regulating adenosine-to-inosine editing, cassette exon and back splicing, and splicing fidelity. NF45-NF90 complexes recognise dsRNA and, in human cells, primarily interact with inverted Alu repeats (AluIRs) that are commonly inserted into introns and other non-coding RNA regions. Intronic AluIRs of [~]300 bp can regulate splicing outcomes, such as generation of circRNAs. We examined domain reorganisation of NF45-NF90 domains on dsRNAs exceeding 50 bp to gain insight into its RNA recognition properties on longer dsRNAs. Using a combination of phylogenetic analysis, solution methods (including small angle X-ray scattering and quantitative cross-linking mass spectrometry), machine learning and negative stain electron microscopy, we generated a model of NF45-NF90 complex formation on dsRNA. Our data reveal that different interactions of NF45-NF90 complexes allow these proteins to coat long stretches of dsRNA. This property of the NF45-NF90 complex has important implications for how long, nuclear dsRNAs are recognised in the nucleus and how this might promote (co)-regulation of specific RNA splicing and editing events that shape the mammalian transcriptome.

biochemistry↗

bioRxiv2022

Structural analysis of SALL4 zinc-finger domain reveals a link between AT-rich DNA binding and Okihiro syndrome

Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.

biochemistry↗

bioRxiv2022

Modulation of RNA binding properties of the RNA helicase UPF1 by its activator UPF2

The NMD helicase UPF1 is a prototype of the superfamily 1 (SF1) of RNA helicases that bind RNA with high affinity and translocate on it in an ATP-dependent manner. Previous studies showed that UPF1 has a low basal catalytic activity that is greatly enhanced upon binding of its interaction partner, UPF2. Activation of UPF1 by UPF2 entails a large conformational change that switches the helicase from an RNA-clamping mode to an RNA-unwinding mode. The ability of UPF1 to bind RNA was expected to be unaffected by this activation mechanism. Here we show, using a combination of biochemical and biophysical methods, that binding of UPF2 to UPF1 drastically reduces the affinity of UPF1 for RNA, leading to a release of the bound RNA. Although UPF2 is capable of binding RNA in vitro, our results suggest that dissociation of the UPF1-RNA complex is not a consequence of direct competition in RNA binding but rather an allosteric effect that is likely mediated by the conformational change induced by binding of the helicase to its activator. We discuss these results in light of transient interactions forged during mRNP assembly, particularly in the UPF1-dependent mRNA decay pathways.

biochemistry↗

bioRxiv2020

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

The conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 [A] resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

biochemistry↗