Search

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues.

Cleavage factor Im (CF Im) is required for the first step in pre-mRNA 3′-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68- kDa subunits. The binding of CF Im to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF Im involved in RNA binding, protein-protein interactions, and subcellular localization. CF Im68 has a modular domain organization consisting of an N-terminal RNA recognition motif and a C-terminal alternating charge domain. However, the RNA recognition motif of CF Im68 on its own is not sufficient to bind RNA but is necessary for association with the 25-kDa subunit. RNA binding appears to require a CF Im68/25 heterodimer. Whereas multiple protein interactions with other 3′-end-processing factors are detected with CF Im25, CF Im68 interacts with SRp20, 9G8, and hTra2β, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF Im68 to the nucleus. However, CF Im68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found. [ABSTRACT FROM AUTHOR]

Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a null background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence. [ABSTRACT FROM AUTHOR]

Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3' end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3' end processing complex, CF I(m)68, stimulates mRNA export. CF I(m)68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF I(m)68 may act as a novel adaptor for NXF1/TAP, we show that CF I(m)68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF I(m)68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3' end processing factor CF I(m)68 in mRNA export.