Hélène Jacquemin-Sablon

A Mechanism for Translationally Coupled mRNA Turnover: Interaction between the Poly(A) Tail and a c-fos RNA Coding Determinant via a Protein Complex

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA‐binding affinity

mRNP3 and mRNP4 (also called FRGY2) are two mRNA‐binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3–4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3–4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3–4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3–4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.

Effect of proteolysis on the yeast mitochondrial deoxyribonucleases

Abstract One or two forms of deoxyribonuclease may be found when yeast mitochondrial membranes are extracted by treatment with Triton X-100 at high ionic strength. The two forms can be separated by chromatography on DEAE-cellulose. The forms differ from one another by their physical and catalytic properties: form I, retained on the column, degrades double-stranded and single-stranded DNA at neutral pH, and is not stimulated by ethidium bromide; form II (or ethidium bromide-activated DNAase), not retained on the column. degrades ss-DNA at neutral pH, and ds-DNA at pH 5.8. Form II is about 30-fold stimulated by ethidium bromide at pH 7.8. Most probably, form II derived from form I through a limited proteolytic process. This conversion depends on such factors as the storage of the mitochondrial extract or the physiological state of the cells. Form II is found when cells are harvested at stionary phase, coincident with the increment of the yeast proteinases; its formation is blocked by the ddition of proteinase inhibitors to the extract. Conversion of form I to form II, which is associated with a change in the sedimentation coefficient of the enzumatic proteins from 5.4 S to 4.5 s, can be reproduced in vitro by treatment with α-chymotrypsin.