No abstract

Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3′ end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3′ end processing regulation by proposing several overlapping models of regulation.

In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K-AKT-mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E-eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E-eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

G-quadruplexes are noncanonical structures formed by G-rich DNA and RNA sequences that fold into a four-stranded conformation. Experimental studies and computational predictions show that RNA G-quadruplexes are present in transcripts associated with telomeres, in noncoding sequences of primary transcripts and within mature transcripts. RNA G-quadruplexes at these specific locations play important roles in key cellular functions, including telomere homeostasis and gene expression. Indeed, RNA G-quadruplexes appear as important regulators of pre-mRNA processing (splicing and polyadenylation), RNA turnover, mRNA targeting and translation. The regulatory mechanisms controlled by RNA G-quadruplexes involve the binding of protein factors that modulate G-quadruplex conformation and/or serve as a bridge to recruit additional protein regulators. In this review, we summarize the current knowledge on the role of G-quadruplexes in RNA biology with particular emphasis on the molecular mechanisms underlying their specific function in RNA metabolism occurring in physiological or pathological conditions.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.

The human proto-oncogene c-myc encodes two proteins, c-Myc1 and c-Myc2, from two initiation codons, CUG and AUG, respectively. It is also transcribed from four alternative promoters (P0, P1, P2, and P3), giving rise to different RNA 5-leader sequences, the long sizes of which suggest that they must be inefficiently translated by the classical ribosome scanning mechanism. Here we have examined the influence of three c-myc mRNA 5-leaders on the translation of chimeric myc-CAT mRNAs. We observed that in the reticulocyte rabbit lysate, these 5-leaders lead to cap-independent translation initiation. To determine whether this kind of initiation resulted from the presence of an internal ribosome entry site (IRES), COS-7 cells were transfected with bicistronic vectors containing the different c-myc 5-leaders in the intercistronic region. An IRES was identified, requiring elements located within the P2 leader, between nucleotides ؊363 and ؊94 upstream from the CUG start codon. This is the first demonstration of the existence of IRES-dependent translation for a proto-oncogene. This IRES could be a translation enhancer, allowing activation of c-myc expression under the control of trans-acting factors and in response to specific cell stimuli.

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miR-NA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17β-estradiol (E 2 ). We show that the expression of a broad set of miRNAs decreases following E 2 treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E 2 -dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E 2 -repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer. [Cancer Res 2009;69(21):8332-40]

The use of several translation initiation codons in a single mRNA, by expressing several proteins from a single gene, contributes to the generation of protein diversity. A small, yet growing, number of mammalian mRNAs initiate translation from a non-AUG codon, in addition to initiating at a downstream in-frame AUG codon. Translation initiation on such mRNAs results in the synthesis of proteins harbouring different amino terminal domains potentially conferring on these isoforms distinct functions. Use of non-AUG codons appears to be governed by several features, including the sequence context and the secondary structure surrounding the codon. Selection of the downstream initiation codon can occur by leaky scanning of the 43S ribosomal subunit, internal entry of ribosome or ribosomal shunting. The biological significance of non-AUG alternative initiation is demonstrated by the different subcellular localisations and/or distinct biological functions of the isoforms translated from the single mRNA as illustrated by the two main angiogenic factor genes encoding the fibroblast growth factor 2 (FGF2) and the vascular endothelial growth factor (VEGF). Consequently, the regulation of alternative initiation of translation might have a crucial role for the biological function of the gene product.