KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.

In Vivo and In Vitro Characterization of the RNA Binding Capacity of SETD1A (KMT2F).

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two-SETD1A and SETD1B-contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs.

Deep structural insights into RNA-binding disordered protein regions.

Recent efforts to identify RNA binding proteins in various organisms and cellular contexts have yielded a large collection of proteins that are capable of RNA binding in the absence of conventional RNA recognition domains. Many of the recently identified RNA interaction motifs fall into intrinsically disordered protein regions (IDRs). While the recognition mode and specificity of globular RNA binding elements have been thoroughly investigated and described, much less is known about the way IDRs can recognize their RNA partners. Our aim was to summarize the current state of structural knowledge on the RNA binding modes of disordered protein regions and to propose a classification system based on their sequential and structural properties. Through a detailed structural analysis of the complexes that contain disordered protein regions binding to RNA, we found two major binding modes that represent different recognition strategies and, most likely, functions. We compared these examples with DNA binding disordered proteins and found key differences stemming from the nucleic acids as well as similar binding strategies, implying a broader substrate acceptance by these proteins. Due to the very limited number of known structures, we integrated molecular dynamics simulations in our study, whose results support the proposed structural preferences of specific RNA-binding IDRs. To broaden the scope of our review, we included a brief analysis of RNA-binding small molecules and compared their structural characteristics and RNA recognition strategies to the RNA-binding IDRs. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.

PhaSePro: the database of proteins driving liquid-liquid phase separation.

Membraneless organelles (MOs) are dynamic liquid condensates that host a variety of specific cellular processes, such as ribosome biogenesis or RNA degradation. MOs form through liquid-liquid phase separation (LLPS), a process that relies on multivalent weak interactions of the constituent proteins and other macromolecules. Since the first discoveries of certain proteins being able to drive LLPS, it emerged as a general mechanism for the effective organization of cellular space that is exploited in all kingdoms of life. While numerous experimental studies report novel cases, the computational identification of LLPS drivers is lagging behind, and many open questions remain about the sequence determinants, composition, regulation and biological relevance of the resulting condensates. Our limited ability to overcome these issues is largely due to the lack of a dedicated LLPS database. Therefore, here we introduce PhaSePro (https://phasepro.elte.hu), an openly accessible, comprehensive, manually curated database of experimentally validated LLPS driver proteins/protein regions. It not only provides a wealth of information on such systems, but improves the standardization of data by introducing novel LLPS-specific controlled vocabularies. PhaSePro can be accessed through an appealing, user-friendly interface and thus has definite potential to become the central resource in this dynamically developing field.

Disordered Regions of Mixed Lineage Leukemia 4 (MLL4) Protein Are Capable of RNA Binding.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.