A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

Abstract

The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

Operation of a TCA cycle subnetwork in the mammalian nucleus

“…However, aside from the specific case of succinylation of histone proteins ( 22 ), succinylation of nuclear proteins has been detected in whole-cell lysates without information about their localization. The charge and modification state of lysine residues may affect the RNA interactions in canonical RNA binding or protein localizations ( 44 ). Succinylation inverts the charge of lysine residues and adds a bulky adduct, therefore imposing a significant functional impact ( 45 , 46 ) on the proteins.…”

Modulated termination of non-coding transcription partakes in the regulation of gene expression

“…For instance, it has been shown that several stress-responsive genes can be repressed by premature termination by the NNS-complex and alternative TSS usage under stress conditions allows excluding Nrd1 and Nab3 recognition sequences from the 5′ UTR and, thus, escaping NNS binding and transcription termination ( 55 ). In addition, several phosphorylation sites have been identified in Nrd1 ( 56 ) and very recently several methylation sites have been detected in both Nrd1 and Nab3 ( 57 ), but a role for these modifications in the regulation of transcription termination has not been demonstrated. The levels of the Sen1 protein have shown to be modulated during the cell cycle ( 58 ).…”

Using Machine Learning and Targeted Mass Spectrometry to Explore the Methyl-Lys Proteome

“…PRM-MS can differentiate and relatively quantify peptides with isobaric post-translational modifications (PTMs). In yeast, in which the histone H3 K4 mono-methyltransferase SET1 was lacking, PRM-MS was used to quantify several newly identified methyl modifications ( Lee et al., 2020 ). Building a PRM-MS isolation list.…”

“… (B) Relative total peak area of Nab3-K363me1 chromatographs in WT versus set1Δ , set2Δ , or set3Δ strains. Figure modified from Lee et al. (2020) .…”