MYND-less splice variants of AML1-MTG8 (RUNX1-CBFA2T1) are expressed in leukemia with t(8;21).

The AML1-MTG8 fusion gene is generated by chromosome translocation t(8;21), which is frequently observed in acute myeloid leukemia. The fusion gene produces a chimeric transcription factor that suppresses the expression of AML1-target genes via the MTG8 part of the chimeric protein, which is thought to be the primary cause of leukemia. The C-terminal region of MTG8 contains the MYND domain, represented by highly conserved zinc-finger-like protein motifs, and is known to interact with corepressor proteins. We found that, instead of the MYND domain, an alternative last exon of MTG8 encoding 27 amino acids in-frame is expressed naturally in human adult testis and in several leukemia cell lines. This type of alternative splicing also occurred in the AML1-MTG8 fusion gene at high levels in leukemia cell lines with t(8;21), as well as in blast cells of leukemia patients with t(8;21). The variant proteins of both MTG8 and AML1-MTG8 reduced transcriptional repressor activity in a mammalian two-hybrid assay. However, mixed expression of these variants with wild-type MTG8 recovered their repressor activity, suggesting that these variants also act as repressors in vivo where wild-type MTG8 and other family members exist in abundance. On the other hand, the MYND-less variants acquired a higher affinity for binding to MTG8 and formed a multimer, whereas the wild-type protein forms a dimer. Thus, expression of the MYND-less variants by the dysregulation of splicing machinery, which stimulates the oligomerization of fusion proteins in leukemia cells, may enhance malignant conversion of hematopoietic cells.

Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly.

Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.

Ribosome recycling factor disassembles the post-termination ribosomal complex independent of the ribosomal translocase activity of elongation factor G.

Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.

Transient idling of posttermination ribosomes ready to reinitiate protein synthesis.

The fate of ribosomes between termination and initiation during protein synthesis is very basic, yet poorly understood. Here we found that translational reinitiation of the alkaline phosphatase gene occurs in Escherichia coli from an internal methionine codon when the authentic translation is prematurely terminated at a nonsense codon that is within seven codons upstream of the reinitiation codon (which we refer to as "reinitiation window"). Changing the reading frame downstream of the stop codon did not abolish the reinitiation, while inactivating the upstream initiation codon abolished the reinitiation. Moreover, depletion of the ribosome recycling factor (RRF), which disassembles posttermination ribosomes in conjunction with elongation factor G, did not influence the observed reinitiation. These findings suggest that posttermination ribosomes can undergo a transient idling state ready to reinitiate protein synthesis even in the absence of the Shine-Dalgarno (SD) sequence within the reinitiation window by evading disengagement from the mRNA.