Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing

Abstract

In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID-MS, to identify binding partners for nuclear actin. Common highconfidence interactions highlight the role of actin in chromatin remodeling complexes and identify the hATAC histone modifier as a novel actin-containing nuclear complex. Further analysis demonstrates that actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. BioID-MS, which can detect also transient interactions, links actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative exon skipping of the SMN2 minigene, suggesting also a functional role for actin in RNA splicing. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.

Nuclear actin dynamics in gene expression and genome organization

“…Recently, our lab utilized two complementary mass spectrometry (MS) techniques, affinity-purification (AP) AP-MS and BioID to identify both stable and dynamic interactions of nuclear actin, and demonstrated that actin is a subunit of the human Ada-Two-Acontaining (hATAC) histone modifier complex. Actin binds directly to KAT14, one of the histone acetyl transferases (HATs) in this complex, and can modify its activity both in vitro and in vivo [54]. Actin may also control histone acetylation via its interactions with histone deacetylases HDAC1 and 2.…”

“…Binding of actin to both promoters and gene bodies [74] suggests that actin could have a role during several steps of the transcription process. Indeed, actin has been linked both to pre-initiation complex (PIC) formation [78], and to transcription elongation [54,[79][80][81]. During the latter process, established interactions with actin monomers take place.…”

“…Thereby actin could have a role in the pause-release of Pol II, where Pol II pauses in the promoter-proximal region and requires P-TEFb to phosphorylate serine 2 in Pol II Cterminal domain (CTD) in order to continue productive transcription [82]. Indeed, analysis of splice site selection in nuclear actin manipulated cells has indirectly suggested a role for actin in controlling transcription elongation rate [54]. Moreover, actin, likely in its monomeric form, interacts with heterogeneous nuclear ribonucleoprotein U (hnRNP U) and this interaction plays a role in transcription elongation [80].…”

Cytoskeletal proteins in the cell nucleus: a special nuclear actin perspective

“…Some studies, however, have suggested a potential role for the RNP-associated actin in facilitating RNP assembly and disassembly at translation sites (Kotani et al , 2013). The wide role of actin is also supported by a recent mass spectrometry–based analysis of nuclear actin-interacting proteins, which revealed dynamic association of actin with proteins implicated in transcription preinitiation, elongation, and pre-mRNA processing (Viita et al , 2019). In addition, minigene splicing assays demonstrated a functional role for actin in alternative splicing, most likely by influencing the rate of transcription elongation (Viita et al , 2019).…”

“…The wide role of actin is also supported by a recent mass spectrometry–based analysis of nuclear actin-interacting proteins, which revealed dynamic association of actin with proteins implicated in transcription preinitiation, elongation, and pre-mRNA processing (Viita et al , 2019). In addition, minigene splicing assays demonstrated a functional role for actin in alternative splicing, most likely by influencing the rate of transcription elongation (Viita et al , 2019). However, the molecular details are still unknown, and await high-resolution structures of actin in complex with the relevant proteins.…”