Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation.

MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domain-containing chromatin regulators that are involved in the regulation of transcriptional "memory" during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5(tm1Apa)) in the murine Mll5 locus. Unlike other Mll genes, Mll5(tm1Apa) homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5(tm1Apa) homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cell-autonomous impairment of neutrophil function. Second, Mll5(tm1Apa/tm1Apa) mice exhibit a mild impairment of erythropoiesis. Third, Mll5(tm1Apa/tm1Apa) hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to self-renewal stimulation by NUP98-HOXA10. Fourth, Mll5(tm1Apa) homozygous HSCs show a dramatic sensitivity to DNA demethylation-induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis.

Identification of c-myc coding region determinant RNA sequences and structures cleaved by an RNase1-like endoribonuclease.

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.

Destroying RNA as a therapeutic approach.

The ability to target RNA, mRNA and viral RNA in particular, for degradation is a powerful approach in molecular biology and pharmacology. Such approaches can be used in the study of gene function as in functional genomics, in the identification of disease-associated genes, and for the treatment of human diseases. This review provides a comprehensive up-to-date look at all the current available technologies used for the destruction of RNA, with a focus on their therapeutic potential. This includes approaches that utilize the activity of protein ribonucleases such as antisense oligonucleotide, small interfering RNA, RNase P-associated external guide sequence, onconase and bovine seminal RNase. Sequence-specific approaches that do not utilize activity of protein ribonucleases, such as ribozyme and DNazyme, are also reviewed and discussed. This review should provide a useful starting framework for researchers interested in using the RNA-destruction methodologies on the bench and in the clinic, and serves as a stimulus for further development of novel and more potent RNA degradation technologies. This is particularly critical, given the anticipation of discoveries of new cellular RNA degradation machineries and human diseases that are associated with dysfunctional RNA molecules.

Purification and characterization of a novel mammalian endoribonuclease.

Endonuclease-mediated mRNA decay appears to be a common mode of mRNA degradation in mammalian cells, but yet only a few mRNA endonucleases have been described. Here, we report the existence of a second mammalian endonuclease that is capable of cleaving c-myc mRNA within the coding region in vitro. This study describes the partial purification and biochemical characterization of this enzyme. Five major proteins of approximately 10-35 kDa size co-purified with the endonuclease activity, a finding supported by gel filtration and glycerol gradient centrifugation analysis. The enzyme is an RNA-specific endonuclease that degrades single-stranded RNA, but not double-stranded RNA, DNA or DNA-RNA duplexes. It preferentially cleaves RNA in between the pyrimidine and purine dinucleotides UA, UG, and CA, at the coding region determinant (CRD) of c-myc RNA. The enzyme generates products with a 3'hydroxyl group, and it appears to be a protein-only endonuclease. It does not possess RNase A-like activity. The enzyme is capable of cleaving RNAs other than c-myc CRD RNA in vitro. It is Mg(2+)-independent and is resistant to EDTA. The endonuclease is inactivated at and above 70 degrees C. These properties distinguished the enzyme from other previously described vertebrate endonucleases.