Speakers – Jan 2017

DNMT3B DNA and RNA interactions

Hong Kee Tan
Tenen Laboratory
Cancer Science Institute of Singapore
National University of Singapore

DNMT3B is known to bind both DNA and RNA in vivo. However, the functions DNMT3B- DNA and RNA interactions are not well studied up to date. Recently, DNMT3B had been shown to interact with ribosomal DNA (rDNA) promoter RNA and regulating rDNA silencing. However, a global mapping of DNMT3B-interacting-RNAs (3DiRNAs) has never been reported up to date. By using DNMT3B RNA immunoprecipitation sequencing (RIP-seq), we mapped the global 3DiRNAs in H1 human embryonic stem cell (ESC) for the first time. Our analysis showed that DNMT3B interacts with nascent transcripts rather than promoter RNAs, favouring to interact with long intron transcripts. 3DiRNAs were found dominating by intronic RNA rather than exonic RNA . In addition, 3DiRNAs showed the strong 5’ end bias in the transcripts. In contrast, we found that DNMT3B-DNA interaction mainly occurs after the first splicing junction where the DNMT3B-RNA interaction signals diminished. In addition, we found that the binding of DNMT3B highly correlated to CpA methylation found in H1 human ESC. Nascent RNA interacting protein and CpA methylation has been proposed for RNA splicing regulation. We asked whether DNMT3B can regulate RNA splicing through these novel properties found in human ESC. However, we found no significant change in global gene splicing profile in DNMT3B knock-down cell. This suggests that DNMT3B may not regulate RNA-splicing in human ESC. The biological functions of DNMT3B-DNA and RNA interactions in hESC are remained unknown at the moment.

A mechanism underlying PTBP1-mediated splicing activation

Fursham Hamid
Makeyev Laboratory
Nanyang Technological University

The outcome of alternative pre-mRNA splicing (AS) events is, in part, regulated by RNA-binding proteins (RBPs) that function as activators or repressors of splicing. An emerging theme in this field of research is that some splicing regulators carry out dual splicing functions depending on its binding position relative to the target exon. One such RBP is Polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I), a well-studied global repressor of neuron-specific alternative splicing program in non-neuronal cells. Ptbp1 induces splicing repression when bound to region upstream of a 5’ splice site but functions as an activator when localized downstream of a 5’ splice site. While the mechanisms underlying Ptbp1 repressive function have been well-documented, how exactly this RBP activate splicing is poorly understood. Here, we identified a Ptbp1-activated cassette exon found on the Deltex2 (Dtx2) gene which contained highly conserved pyrimidine-rich tracts exclusively downstream of its 5’ splice site. We found that binding of Ptbp1 onto these motifs recruits U1 snRNP to the 5’ splice by interacting with a specific structural element in the U1 snRNA. Interestingly, insertion of Ptbp1 binding sequences directly upstream of Dtx2 exon 6 was sufficient to repress exon inclusion in a Ptbp1-dependent manner.

Investigating the role of specialized ribosomes in erythroid differentiation

Jie Min Nah
Guo Laboratory
Institute of Molecular and Cell Biology, A*STAR

Ribosomes are made up of ~80 ribosomal proteins (RPs) and 4 ribosomal RNAs (rRNAs), and are traditionally thought to be invariant. Recent studies have suggested the presence of ‘specialised ribosomes’, which can be heterogeneous in their RP composition. These studies have suggested that specialised ribosomes could have important functions in regulating gene expression under different cellular contexts. Ribosomopathies are a group of diseases in which there is defective ribosome biogenesis/function. The most common ribosomopathy in humans is Diamond-Blackfan anemia (DBA) – where a haploinsufficiency of specific RPs leads to a disruption in normal erythropoiesis. Despite the possible connection to ribosomopathies, there is yet any extensive study done to examine the presence of specialised ribosomes in humans.

Our study investigates the role of specialised ribosomes in regulating the erythroid compartment. We hypothesise that ribosomes with specific RPs may participate in driving erythroid differentiation by translating distinct sets of mRNAs. This could potentially explain why RP haploinsufficiency, such as in DBA, may lead to defective erythropoiesis. Using large-scale in vitro differentiation of primary CD34+ cells, we follow cellular transitions through different stages of erythropoiesis. We found that global translation slows at the later stages of erythropoiesis. Interestingly, proteomics analysis of the polysomal proteins showed that levels of RPs change in differing magnitudes as the cells differentiate. This implies that the levels of RPs are not coordinately regulated in a fixed stoichiometry, hence lending support to the idea that heterogeneous ribosomes could be present during erythropoiesis.

We plan to validate the proteomics results and investigate the role of these candidate RPs in erythroid differentiation. To further study how changes in RP expression affects translation, we plan to probe mRNAs translated by ribosomes containing these candidate RPs. Through these investigations, we hope to explore another possible layer of translation control in human gene expression.