Georges Huez

Release of messenger RNA from rabbit reticulocyte polyribosomes at low concentration of divalent cations

Abstract 1. 1. An RNA that we identify as the 9-S messenger RNA previously studied can be released from rabbit reticulocyte polyribosomes by EDTA or CM-cellulose treatment. 2. 2. The criteria for this identification are: high specific radioactivity as compared with ribosomal RNA; high sensitivity to ribonuclease when polyribosomes are treated with minute quantities of this enzyme; a sedimentation velocity identical to that of 9-S messenger RNA after sodium dodecyl sulfate treatment. 3. 3. However, this RNA liberated from reticulocyte polyribosomes by EDTA or CM-cellulose treatment is probably associated with proteins. Indeed, it sediments more rapidly than purified 9-S mRNA if sodium dodecyl sulfate treatment is omitted. 4. 4. 5-S RNA is also released from polyribosomes by the procedure used to liberate messenger RNA.

Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

TIAR and TIA-1 are two closely related RNA-binding proteins which possess three RNA recognition motifs (RRMs) followed by an auxiliary region. These proteins are involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and regulation of mRNA translation. Here we characterize the subcellular localization of these proteins in somatic cells. We demonstrate that TIAR and TIA-1 continuously shuttle between the cytoplasm and the nucleus and belong to the class of RNA-binding proteins whose nuclear import is transcription-dependent. We identified RRM2 and the first half of the auxiliary region as important determinants for TIAR and TIA-1 nuclear accumulation. In contrast, the nuclear export of TIAR and TIA-1 is mediated by RRM3. Both RRMs contribute to TIAR and TIA-1 nuclear accumulation or export by their RNA-binding capacity. Indeed, whereas mutations of the highly conserved RNP2 or RNP1 peptides in RRM2 redistribute TIAR to the cytoplasm, similar modifications in RRM3 abolish TIAR nuclear export. Moreover, TIAR and TIA-1 nuclear accumulation is a Ran-GTP-dependent pathway, in contrast to its nuclear export which is unaffected by Ran-GTP depletion and which is independent of the major CRM1-exporting pathway. This study demonstrates the importance of TIAR and TIA-1 RNA-binding domains for their subcellular localization and provides the first evidence for distinct functions of TIAR and TIA-1 RRMs.

Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages

The turnover of tumor necrosis factor (TNF) mRNA in permanently transfected macrophages of the RAW 264.7 cell line was studied directly (by Northern blot analysis using a probe specific for TNF) and indirectly (through studies of the turnover of various reporter mRNAs, either containing or lacking the TNF 3 untranslated region (UTR)). The TNF mRNA was found to be very unstable in RAW 264.7 cells. Instability appeared to result from two distinguishable nucleolytic processes. The major degradative process involved was not specific for the TNF 3 UTR of reporter mRNAs, and was inhibited by actinomycin D pretreatment. It appeared to be expressed constitutively, in that cell activation by lipopolysaccharide (LPS) did not modify message stability. When cells were treated with actinomycin D, a minor nucleolytic activity was uncovered. This minor activity was noted to increase with time following LPS activation. It also exhibited specificity, in that reporter mRNAs bearing the 3 UTR of TNF were more susceptible to degradation in the presence of actinomycin D than were constructs lacking the 3 UTR of TNF. Thus, TNF mRNA turnover appears complex, and depends upon at least two separable degradative pathways. The TNF 3 UTR apparently contributes only modestly to the instability of this mRNA under normal conditions.

Mapping of a minimal AU-rich sequence required for lipopolysaccharide-induced binding of a 55-kDa protein on tumor necrosis factor-alpha mRNA.

In monocyte/macrophage cells, the translation of tumor necrosis factor-α (TNF-α) mRNA is tightly controlled. In unstimulated cells, TNF-α mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3′-untranslated region of TNF-α mRNA. Several groups have described the binding of proteins on AU-rich elements (AREs). We have previously reported the binding of two cytosolic protein complexes (1 and 2) to the TNF-α mRNA ARE, one of which (complex 2) is observed only following induction of TNF-α production by LPS. In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, whereas the formation of the LPS-inducible complex 2 requires a minimal sequence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs for their ability to form complex 2. We observed that the ARE derived from granulocyte/macrophage colony-stimulating factor mRNA, which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA, which does not contain the nonanucleotide, is unable to promote the formation of any LPS-induced complex.

Translational control mediated by UA-rich sequences

Several messenger RNAs coding for cytokines, growth factors or oncoproteins contain a conserved UA-rich sequence in their 3 untranslated regions. This sequence, which is composed of several interspersed repeats of the octanucleotide UUAUUUAU, is a key element in the posttranscriptional regulation of these mRNAs. Previously shown to be involved in mRNA destabilization, these UA-rich elements can also strongly influence mRNA translation. A translation inhibition mediated by these sequences has indeed been observed in vitro as well as in frog oocyte or in somatic cells.

Mapping and characterization of the minimal internal ribosome entry segment in the human c- myc mRNA 5′ untranslated region

The human c-myc proto-oncogene is transcribed from four alternative promoters generating transcripts with 5′ untranslated regions of various lengths. These transcripts encode two proteins, c-Myc1 and c-Myc2, from two initiation codons, CUG and AUG, respectively. We and others have previously demonstrated that the region of c-myc transcripts between nucleotides (nt) –363 and –94 upstream from the CUG start codon contained an internal ribosome entry site leading to the cap-independent translation of c-myc open reading frames (ORFs). Here, we mapped a 50-nt sequence (−143 −94), which is sufficient to promote internal translation initiation of c-myc ORFs. Interestingly, this 50-nt element can be further dissected into two segments of 14u2009nt, each capable of activating internal translation initiation. We also demonstrate that this 50-nt element acts as the ribosome landing site from which the preinitiation ribosomal complex scans the mRNA until the CUG or AUG start codons.