An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.     

Destroy this (RNA) message after reading it!

Traditionally, mRNA decay was considered a simple destruction step of mRNA. This view has been challenged in the past years and mRNA decay now appears as an essential step in the regulation of gene expression. We first present a short review of the different reactions involved in mRNA decay, as well as some indications on their cellular location. Then, we describe two processes in which mRNA decay plays an essential role: (1) the mRNA quality control mechanisms that get rid of aberrant mRNAs (nonsensE-mediated decay, non-stop decay, no-go decay); (2) the regulation of mRNA stability through the targeting of specific factors to the mRNA (proteins or small non-coding RNAs).     

Crystallization and characterization of Pumilo: a novel RNA binding protein

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6(3) with unit cell dimensions of a = b = 94.5 A, c = 228.9 A, alpha = beta = 90 degrees, gamma = 120 degrees and diffract to 2.6 A with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of alpha-helical secondary structure.     

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.     

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.     

Translational control of development inC. elegans

Translational control by the 3′untranslated regions (3′UTRs) of mRNAs contributes to important events throughout the development of C. elegans. In oocytes and early embryos, maternal mRNAs are controlled by 3′UTR elements to restrict translation of their protein products to specific blastomeres. Localized translation is probably critical for specifying blastomere identity. In both germline and somatic cells, mRNAs from sex determining genes are translationally repressed by 3′UTR controls. These controls balance the activities that specify male and female cell fates. During larval development, the temporal sequence of cell lineages requires 3′UTR-mediated regulation of heterochronic genes by a small non-protein coding RNA. We review what is known about these translational control mechanisms in C. elegans. This overview illustrates that translational control by 3′UTR elements is a powerful mechanism for regulating the expression of multiple gene products in diverse cell types during development of a multi-cellular animal.