Formation of mRNA 3′ ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3′ ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.

Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis

http://gen677.weebly.com/uploads/8/6/5/7/865764/andersonnucleolus2002.pdf

Directed proteomic analysis of the human nucleolus.

The Exosome: A Conserved Eukaryotic RNA Processing Complex Containing Multiple 3′→5′ Exoribonucleases

https://www.cell.com/cell/pdf/S0092-8674(05)00442-3.pdf

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex

Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A) 1 RNA in the nucleus at 37C120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export. The mechanism of export of mRNA from the nucleus to the cytoplasm is remarkably accommodating in the sense that an enormous variety of primary and secondary RNA structures are compatible with export. The fact that neither the 59methyl cap structure nor the 39poly(A) tail appears altogether essential for export (16) suggests that there is a mechanism in the nucleus which allows for RNA recognition independent of the sequence. It is likely that during or immediately after their synthesis, transcripts are quickly packaged into ribonucleoprotein particles (RNP) which enable them to gain access to the periphery of the nucleoplasm and interact specifically with componentsofnuclearpores,throughwhichtheysubsequently translocate (14, 42). If this itinerary is relatively uniform for most mRNAs, it is reasonable to postulate that there are factors which serve as RNA chaperones and/or normalize RNA secondary structure. Single-stranded RNAs spontaneously acquire an extensive secondary structure, which is subject to proteins with annealing activity (p53 and heterogeneous nuclearRNP[hnRNP]A1[24,25])andmeltingactivity(helicases [34]). Studies of the yeastSaccharomyces cerevisiaehave identified several proteins which, when mutated, lead to nuclear accumulation of poly(A) 1 RNA without inhibiting pre-mRNA splicing.Apartfromnucleoporins(8),theseincludescomponents of a nucleocytoplasmic GTPase cycle [the Cnr1/2p (Gsp1/2p) GTPases, Mtr1p (Prp20p), and Rna1p (6, 35)], a cytoplasmic protein (Mtr7p [34a]), the nucleolar protein Mtr3p (19), the nuclearproteinsMtr2p(19)andRat1p(1),andthenuclearhnRNP

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

Nucleocytoplasmic transport of mRNA is vital to gene expression and may prove to be key to its regulation. Genetic approaches in Saccharomyces cerevisiae have led to the identification of conditional mutants defective in mRNA transport. Mutations in approximately two dozen genes result in accumulation of transcripts, trapped at various sites in the nucleus, as detected by in situ hybridization. Phenotypic and molecular analyses of many of these mRNA transport mutants suggest that, in yeast, the function of the nucleus is not limited to the biogenesis of pre-ribosomes but may also be important for transport of poly(A)+ RNA. A similar function of the animal cell nucleolus is suggested by several observations.

mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

Essentially all eukaryotic cells, including murine lymphomas, express surface proteins, such as Thy-1, which are anchored by a phosphoinositol mannolipid. Putative mannolipid anchor precursors can be detected in these cells. Six distinct Thy-1-negative lymphoma mutants lack complete mannolipids, and three mutants synthesize atypical mannolipids. The absence of complete mannolipids can account for the lack of expression of multiple mannolipid-anchored proteins and may also account for the lack of lipid anchoring in the human disease paroxysmal nocturnal hemoglobinuria. Structural information on the mannolipids of wild-type and mutant cells indicates that anchor biosynthesis in these cells may involve both transmembrane flip-flop of intermediates and a deacylation step.

Atypical mannolipids characterize Thy-1-negative lymphoma mutants.

The addition of glycophospholipid (GPL) anchors to certain membrane proteins occurs in the rough endoplasmic reticulum and is essential for transport of the proteins to the plasma membrane. Limited circumstantial evidence suggests that dolichol-phosphoryl-mannose (DPM) is a donor of mannose residues of these anchors. We here report studies of a CHO cell mutant (B421) transfected to express the GPL-anchored protein, placental alkaline phosphatase (AP). Only a few transfectants were found to express GPL-anchored AP on their surface, and these clones synthesized DPM. Moreover, and most strikingly, when surface AP-negative transfectants were treated with tunicamycin to cause accumulation of DPM, these cells expressed lipid-anchored AP. Fusion of a cloned surface AP-negative transfectant of B421 with the Thy-1-class E mutant thymoma, which is also deficient in DPM synthesis, produced hybrids that synthesized DPM and expressed AP and Thy-1. Thus, two mutations can interrupt DPM synthesis, and three sets of observations point to an essential role of DPM for addition of GPL anchors.

Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin.

A M Tartakoff

Papers

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

Atypical mannolipids characterize Thy-1-negative lymphoma mutants.

Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin.