Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Abstract

Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3' end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3' end of histone mRNA.

Dual role for the RNA-binding domain of Xenopus laevis SLBP1 in histone pre-mRNA processing

“…We also compared the relative affinities of the SLBP in extracts from oocytes expressing the same proteins+ The level of overexpression of SLBP active in RNA binding is high in all of these experiments and is clearly seen by comparing lanes 2 and 9 of Figure 4C with lanes 3 and 10, consistent with the results obtained from the western blot+ Extracts were prepared from the oocytes and the affinity of the proteins for the stemloop measured by the amount of competitor RNA necessary to compete formation of the stem-loop RNA complex+ Because the concentrations of xSLBP1 and 1-2-1 are similar in the two extracts, this provides an estimate of the relative affinities of the two proteins under relatively physiological conditions in the presence of the total proteins from Xenopus+ Each of the proteins also bound the stem-loop with a similar affinity by this criterion (Fig+ 4C)+ To further demonstrate the similar affinity of 1-2-1 and xSLBP1 for the stem-loop, three proteins, myctagged xSLBP1 (which is larger than xSLBP1 and can be distinguished from 1-2-1), 1-2-1, and a 94 amino acid protein 0-1-(1/2), were overexpressed in the same oocyte by injecting a mixture containing equal amounts of the three synthetic mRNAs+ When these three proteins were expressed simultaneously in oocytes, three distinct complexes were formed with the stem-loop RNA in oocyte extracts (Fig+ 4D)+ To assess the relative affinity of the three proteins, mobility shift assays were done using varying amounts of radiolabeled probe+ At low probe concentrations, the three proteins are competing for limiting probe+ At the three concentrations of the probe used, all three proteins formed complexes with the probe+ The complexes are present in similar relative proportions ( Fig+ 4D, lanes 1-3), indicating that all three proteins bound the probe with similar affinity+ If there were any slight differences in affinity, the 1-2-1 and 0-1-(1/2) proteins had a slightly higher affinity than the myc-SLBP1 protein, because at the lowest probe concentrations ( Fig+ 4D, lanes 2 and 3), these complexes were overrepresented compared with the amount of complex formed by the full-length xSLBP1+ Using similar approaches we have previously shown that the full-length endogenous xSLBP1 and xSLBP2 proteins have similar affinities for the stem-loop (Wang et al+, 1999)+ Both the 1-2-1 protein and xSLBP1 (1-1-1) form very stable complexes with the stem-loop RNA+ When the extracts from oocytes overexpressing these two proteins were incubated with the probe for 20 min followed by addition of excess unlabeled competitor RNA, the unlabeled competitor did not displace the pre-bound radiolabeled probe (Fig+ 4E, lanes 3 and 5)+ In contrast, addition of the same amount of unlabeled probe together with the labeled probe completely prevented formation of the labeled complexes (Fig+ 4E, lane 6)+ Thus, like the mammalian SLBP (Williams & Marzluff, 1995), the complexes between the stem-loop and the frog SLBP (and the 1-2-1 protein), are extremely stable+ By all of these criteria, the 1-2-1 protein and the xSLBP1 have similar affinities for the stem-loop+…”

“…When the mouse histone H2a-614 gene is injected into frog oocyte nuclei, properly processed histone mRNA is produced and the mature histone mRNA is efficiently transported to the cytoplasm (Williams et al+, 1994)+ We developed an assay that detects both processed and unprocessed histone mRNAs generated from the injected mouse H2a-614 gene (Wang et al+, 1999), allowing us to determine the efficiency of histone premRNA processing in vivo (Fig+ 1A)+ The 260-nt probe detects processed histone H2a-614 mRNA as a 183-nt fragment+ Any unprocessed histone mRNAs (which would be expected to have heterogeneous 39 ends) will protect a 217-nt fragment (Fig+ 1A)+ Because the probe and each protected fragment has a single radioactive phosphate, the efficiency of processing can be determined by comparing the intensity of the two protected fragments+ Plasmid DNA that is injected into the oocyte cytoplasm is not transcribed, so the results are not affected by the efficiency of injecting the DNA into the nucleus+…”

“…Characterization of the processing of histone pre-mRNA derived from injected DNA+ A: The S1 nuclease protection assay used to measure histone pre-mRNA processing is diagramed+ The 260-nt probe contains 43 nt that are not present in the H2a-614 histone pre-mRNA, but are derived from the vector pBluescript KS (zig-zag line)+ The unprocessed RNAs protect a 217-nt fragment regardless of their 39 end and the processed histone mRNAs protect a 183-nt fragment+ Because the probe is 39-end labeled, the ratio of the intensity of the two fragments is a measure of processing efficiency+ B: Frog oocytes were preincubated in buffer (lane 2) or with 200 mg/mL Actinomycin D (lane 3) for 1 h+ They were then injected with the histone H2a-614 gene and blue Dextran, and incubated in the continued presence of the inhibitor for 18 h+ RNA was prepared and then assayed for histone H2a-614 mRNA+ Lane 1 is marker pUC18 digested with MspI+ C: Oocytes were injected with the H2a-614 gene and blue Dextran, incubated for 18 h and then half of the oocytes were harvested (lane 1) and the other half were incubated for an additional 5 h in 200 mg/mL Actinomycin D (lane 2)+ A separate batch of oocytes was injected with either the H2a-614 gene (lane 3) or with the histone H2a-4G gene (lane 4) and incubated for 18 h+ The H2a-4G gene produces a transcript that cannot be processed because of the insertion of four Gs at the cleavage site )+ RNA was prepared from the oocytes and RNA from the equivalent of one oocyte analyzed by S1 nuclease mapping+ D: Frog oocytes were injected with the histone H2a-614 gene and the levels of processed and unprocessed histone mRNAs measured 18 h later (lane 1)+ In lane 2, the human U7 gene (Jacobs et al+, 1999) was coinjected into the nucleus with the H2a-614 gene+ In lane 3 the oocytes were injected with synthetic xSLBP1 mRNA 30 h prior to injection of the histone H2a-614 gene+ In lane 4, the oocytes were injected with the xSLBP1 mRNA 30 h prior to coinjection of the human U7 gene and the histone H2a-614 gene+ The processing efficiency (percent processing) quantified using a PhosphorImager is indicated below each lane+ xSLBP1 and U7 snRNP cooperate to process histone pre-mRNA in vivo Histone pre-mRNA processing in oocytes requires both xSLBP1 (Wang et al+, 1999) and the U7 snRNP (Scharl & Steitz, 1996) as well as other factors+ A critical step in vitro is assembly of a stable complex containing SLBP and U7 snRNA on the histone pre-mRNA , and efficient assembly of the complex requires both stable binding (high affinity) of SLBP to the stem-loop and base-pairing of U7 snRNP to the pre-mRNA )+ To determine whether xSLBP1 or U7 snRNP were limiting for processing in vivo we increased the concentration of U7 snRNA by expressing U7 snRNA from the human U7 snRNA gene (Jacobs et al+, 1999) and the concentrations of xSLBP1 by injecting synthetic xSLBP1 mRNA+ There are excess snRNP proteins present in the frog oocyte that will assemble U7 snRNA into functional U7 snRNP particles (Strub & Birnstiel, 1986)+ In this experiment we used a batch of oocytes that processed the histone H2a-614 mRNA relatively inefficiently (Fig+ 1D, lane 1)+ Overexpression of either U7 snRNA or xSLBP1 resulted in an increased efficiency of processing (Fig+ 1D, lanes 2 and 3)+ Overexpression of both xSLBP1 and U7 snRNA in the same oocytes resulted in a further increase in processing (Fig+ 1D, lane 4)+ Thus both U7 snRNP and xSLBP1 are present at suboptimal concentrations for processing the histone H2a-614 pre-mRNA expressed in the oocyte+ This result is consistent with the suggestion that these two factors cooperate in histone pre-mRNA processing …”

“…The RBD of SLBP1 and SLBP2 are 70% identical and bind the stem-loop with similar affinity (Wang et al+, 1999)+ To determine if the only role of the RBD in histone pre-mRNA processing was to bind the protein to the pre-mRNA, we tested the ability of the chimeric 1-2-1 SLBP to complement processing+ The same batch of oocytes was injected with a control solution or mRNAs encoding one of the following proteins: xSLBP1, Xen- Because histone pre-mRNA processing occurs in the nucleus, we tested the ability of 1-2-1 to efficiently localize to the nucleus+ The injection of synthetic mRNA encoding the various SLBPs resulted in at least a 10-20-fold increase in active (based on RNA binding) SLBP in the frog oocytes (Wang et al+, 1999; see Fig+ 4C, lanes 2-3 and 9-10)+ Synthetic mRNA encoding the relevant clone was injected into oocytes+ Two days later, oocytes were fractionated into nuclear and cytoplasmic components and the fractions assayed by the mobility shift assay+ As we have previously shown (Wang et al+, 1999), overexpressed SLBP1 is found in both the nucleus and the cytoplasm in similar proportions as the endogenous SLBP1, and overexpressed xSLBP2 is Xenopus oocytes were injected with synthetic mRNAs encoding the indicated RNA-binding proteins+ Forty-eight hours later the oocyte nuclei were injected with a plasmid encoding the mouse histone H2a-614 gene+ Eighteen hours later RNA was prepared and assayed for processed (Proc) and unprocessed (Unproc) mouse histone H2a-614 mRNA using the S1 nuclease assay diagramed in Figure 1A+ Lane labeled M is pUC18 DNA digested with MspI+ The results are representative of at least three independent experiments with each construct+ A: Oocytes were injected with buffer (lane 2), synthetic mRNA encoding Xenopus HnRNP A1 (lane 3), xSLBP1 (lane 4), or the 1-2-1 chimeric protein (lane 5)+ The percent processing determined using a PhosphorImager is indicated below each lane+ B: Oocytes were injected with buffer (lane 1), synthetic mRNA encoding xSLBP2 (lane 2), xSLBP1 (lane 3), or the chimeric protein 1-2-1 (lane 4) and assayed for the processing of the H2a-614 pre-mRNA+ The processing efficiency (percent processing) quantified using a PhosphorImager is indicated below each lane+ C: Oocytes were injected with synthetic mRNA encoding the indicated protein+ Forty-eight hours later the oocytes were fractionated into cytoplasmic and nuclear fractions (C and N), and the localization of the proteins determined using the mobility shift assay+ Each lane represents material from one oocyte+ Only the complexes formed (designated xSLBP1/RNA and xSLBP2/RNA) are shown; the unbound probe is not shown in this experiment+ Lanes 1 and 2: xSLBP1; lanes 3 and 4: 1-2-1; lanes 5 and 6: xSLBP2+…”

“…In mammalian cells, SLBP functions in the processing reaction in the nucleus and accompanies the histone mRNA to the cytoplasm, where it remains with the mRNA on polyribosomes as a component of the histone mRNP (Hanson et al+, 1996)+ Recently we have cloned two SLBPs from frog oocytes (Wang et al+, 1999)+ xSLBP1 is the homolog of the mammalian SLBP and is involved in histone pre-mRNA processing+ The other protein, xSLBP2, is cytoplasmic and bound to histone mRNA in the frog oocyte+ During oocyte maturation, xSLBP2 is destroyed and histone mRNA associates with xSLBP1 (Wang et al+, 1999)+ These two SLBPs are similar only in their 73 amino acid RNA-binding domains (RBD) located in the center of each protein (Wang et al+, 1999)+…”