Biology subjects

Rissland, O.

Publications and source records attributed to Rissland, O..

3 recordsLinked to original sources

bioRxiv2024

UBR4 regulates a MetAP2-dependent Arg/N-degron pathway

The open reading frame does more than merely encode a linear peptide sequence; it is a reservoir of regulatory information. Here, as part of investigations into how the N-terminal amino acids regulate translation, we serendipitously uncovered a new N-degron that revealed an additional layer of regulation in these pathways. Using reporter assays, we discovered that peptides bearing position 3 arginine or lysine residues at the N-terminus were rapidly degraded in mammalian cells. We found this pathway requires MetAP2, which co-translationally cleaves the N-terminal methionine preceding second position threonine and valines to initiate protein decay. We used CRISPR-Cas9 to knockout key N-recognins and found that these N-degrons are exclusively targeted by the E3 ligase UBR4, but not by UBR1 or UBR2. Together, our results characterize a new N-degron pathway that reveals a unique role for MetAP2 and UBR4 in mediating protein decay. SIGNIFICANCEThe Arg/N-degron pathway targets position 1 or 2 N-terminal Lys and Arg residues via UBR Box E3 ligases to trigger protein decay. Here we show that UBR4 can specifically recognize position 3 Lys and Arg N-termini upon methionine removal by the methionine amino peptidase MetAP2. Accordingly, proteins that bear N-terminal residues that are processed by MetAP1 are unaffected by the loss or inhibition of MetAP2. Using a combination of reporter assays, and bioinformatic approaches were identified endogenous proteins whose N-termini are recognized by this MetAP2-dependent Arg/N-degron pathway. Thus, our results expand the number of Arg/N-degron substrates and describe a new mechanism through which they are targeted.

molecular biology↗

bioRxiv2024

Muskelin acts as a substrate receptor of the highly regulated Drosophila CTLH E3 ligase during the maternal-to-zygotic transition

The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental specificity of the CTLH complex is mediated by multi-pronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, we find that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.

developmental biology↗

bioRxiv2022

Synonymous codon usage regulates translation initiation

Synonymous codon usage regulates gene expression such that transcripts rich in optimal codons produce significantly more protein than their nonoptimal counterparts. A major unresolved issue has been understanding the mechanisms by which synonymous codons regulate gene expression. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger mRNA degradation. However, differences in transcript abundance are not always sufficient to explain differences in protein levels, suggesting there are additional mechanisms by which codon usage regulates gene expression. Using reporter assays in human and Drosophila cells, we found that transcript levels account for less than half of the variation in protein abundance. We demonstrate that the differences at the protein level are not attributable to either protein folding or stability. Instead, we find that mRNAs with nonoptimal codons are bound by fewer ribosomes and that nonoptimal codon usage represses translation initiation. Nonoptimal transcripts are also less bound by the key translation initiation factors eIF4E and eIF4G, providing a mechanistic explanation for their reduced initiation rates. Our results reveal a new mechanism of regulation by codon usage, where nonoptimal codons repress further rounds of translation.

molecular biology↗