Uncovering the Roles of Human C2H2-Zinc Finger Protein ZNF121 in Gene Regulation

Gene expression is a complex, dynamic, and tightly orchestrated process. Regulation of genes transcribed by RNA polymerase II is coordinated by an elegant arrangement of protein machinery and chromatin architecture, and the resulting pre-mRNA is post-transcriptionally processed and regulated at every step to buffer, reduce, or amplify its protein output. Chemical nucleotide modifications on DNA or RNA are one type of gene regulatory mechanism. N6-methyladenosine (m6A) is the most abundant and well-characterized mRNA modification, facilitating mRNA regulatory processes that include mRNA decay regulated by the m6A reader proteins YTH-domain-containing family protein 2 (YTHDF2). However, YTHDF2 has a relatively weak affinity for its mRNA targets. RNA-binding proteins (RBPs) bind transcripts to exert regulatory effects, and many DNA-binding C2H2-zinc finger proteins (ZFPs) have recently been shown to be RBPs with roles in mRNA biology. I identified C2H2-ZFP, ZNF121, as a YTHDF2 binding partner in affinity purification and mass-spectrometry (AP-MS) experiments and I hypothesized that, like other tested ZFPs, ZNF121 is an RNA-binding protein and acts as a cofactor to stabilize YTHDF2 on shared mRNA targets. In this thesis, I utilize multi-omic and biochemical approaches to understand the role of ZNF121 in gene expression. I observe that ZNF121 is a cyto-nuclear protein and confirm its interaction with YTHDF2 and RNA. Using iCLIPseq, I found that ZNF121 binds mRNA around sites of YTHDF2 enrichment, even when no nearby m6A modification exists. Specifically, over 80% of ZNF121-bound transcripts are also YTHDF2-bound transcripts. Upon depletion of ZNF121, the binding of YTHDF2 to shared transcripts is impaired, and the stability of these transcripts increases in an m6A-independent fashion. This affects many transcripts coding for cell-cycle related proteins like MDM2, with effects on the DNA damage response and cell viability. Lastly, I observe that ZNF121 regulates cassette exon retention in the nucleus. My results show that ZNF121, beyond its conventional role as a transcription factor, is a versatile protein that regulates both mRNA stability in conjunction with YTHDF2 and mRNA splicing.

Department dc:contributor.department

Molecular Genetics

Year dc:date.issued

2025

Author dc:creator

• Burke, Giovanni Livingston

Advisor dc:contributor.advisor

• Greenblatt, Jack F

• C2H2 Zinc Finger Protein
• m6A
• RNA biology
• RNA modifications
• YTHDF2
• ZNF121

Statement dc:rights

• Attribution 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by/4.0/

Handle dc:identifier.uri

https://hdl.handle.net/1807/150378

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/150378