Fuke H and Ohno M (2008), Role of poly (A) tail as an identity element fo...

XB-ART-37356

Nucleic Acids Res 2008 Feb 01;363:1037-49. doi: 10.1093/nar/gkm1120.

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Role of poly (A) tail as an identity element for mRNA nuclear export.

Fuke H , Ohno M .

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Different RNA species are rigorously discriminated and exported by distinct export factors, but this discrimination mechanism remains largely unknown. We previously showed, by RNA microinjection experiments, that intronless mRNAs are discriminated from U snRNAs based on their difference in RNA length. However, it was unclear how they are discriminated in the natural situation in which their nascent transcripts emerge progressively during transcription. We hypothesized that transcription from the corresponding promoters is important for this discrimination. Here we show that contrary to our hypothesis, the discrimination process was not significantly influenced by whether transcription occurred from an mRNA- versus a U snRNA-type promoter. Rather, the features of transcribed RNAs determined the RNA identity, consistent with our previous results of RNA microinjection. Moreover, we found that the poly (A) tail can function as an identity element for mRNA export. The presence of a poly (A) tail of an appropriate length committed otherwise short Pol II transcripts to the mRNA export pathway in a dominant manner, indicating that the poly (A) tail either contributes to increasing the RNA length or functions as a platform to recruit mRNA export factors. Our results reveal a novel function of the poly (A) tail in mRNA export.

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Species referenced: Xenopus laevis
Genes referenced: dhfr mmut phax

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	Figure 1. Effect of RNA length on RNA export in the transcription-coupled system (I). (A) Diagram of the DNA constructs. Human β-globin cDNA fragments of various lengths were inserted into the XhoI site of the U1ΔSm gene. (B) The plasmids harboring the U1ΔSm genes with or without the insertion of cDNA fragments of various lengths (50–300 nt) were microinjected into the nucleus of Xenopus oocytes, with 17 ng/oocyte of either wild-type (WT) or mutant (mut) CTE, and the nucleocytoplasmic distribution of the transcripts was analyzed by northern blotting of nuclear (N) and cytoplasmic (C) RNA fractions after 4 h. The distribution of endogenous U6 snRNA (U6) and 5.8S rRNA (5.8S) was also analyzed by northern blotting to verify the efficacy of the nucleocytoplasmic fractionation. (C) Quantitation of RNA export inhibition by CTE WT from the experiments as in B. Average inhibition and standard deviation were calculated (with CTE mut taken as standard) from three independent experiments. (D) Plasmids harboring the U1ΔSm genes with or without the insertion of the cDNA fragments of 50 or 300 nt were microinjected into the nucleus, with 4.1 ng/oocyte of the PHAXΔNES protein (+) or buffer (−), and export of the transcripts was examined as in B, except that RNA was isolated 3 h after injection. (E) Quantitation of RNA export inhibition by PHAXΔNES from the experiments as in D. Average inhibition and standard deviation were calculated (with buffer taken as standard) from three independent experiments.
	Figure 2. Effect of RNA length on RNA export in the transcription-coupled system (II). (A) Diagram of the DNA constructs. Human β-globin cDNA fragments of various lengths were cloned between the promoter and terminator of the U1 gene. The U1ΔSm gene was also used as a control. (B) Export of the transcripts produced from the microinjected plasmids harboring the cDNA fragments of various lengths (50–360 nt) between the promoter and terminator of the U1 gene was analyzed as in Figure 1B. The U1ΔSm gene was also used as a control. The bands indicated by the asterisk correspond to the 360-nt transcript whose 3′-end was properly formed. (C) Quantitation of RNA export inhibition by CTE WT from the experiments like that shown in B. Since the β360 construct produced multiple bands for unknown reason, the bands near the expected size were quantified (B, asterisk). (D) Export of the transcripts produced from the microinjected plasmids harboring the cDNA fragments of 50 or 360 nt between the promoter and terminator of the U1 gene was analyzed as in Figure 1D. The U1ΔSm gene was also used as a control. The bands indicated by the asterisk correspond to the 360-nt transcript whose 3′-end was properly formed. (E) Quantitation of RNA export inhibition by PHAXΔNES from the experiments like that shown in D. Since the β360 construct produced multiple bands, the bands near the expected size were quantified (D, asterisk). (F) Similar experiments to those in C and E except that cDNA fragments from the DHFR gene instead of the β-globin gene were used.
	Figure 3. Effect of RNA length on RNA export in the transcription-coupled system (III). (A) Diagram of the DNA constructs. One to three copies of a 100-nt DNA fragment from the DHFR gene were cloned between the promoter and terminator of the U1 gene. The U1ΔSm gene was also used as a control. (B) Export of the transcripts produced from the microinjected plasmids harboring 1–3 copies of a 100-nt DNA fragment from the DHFR gene between the promoter and terminator of the U1 gene was analyzed as in Figure 1B. The U1ΔSm gene was also used as a control. (C) Quantitation of RNA export inhibition by CTE WT from the experiments like that shown in B. (D) Export of the transcripts produced from the microinjected plasmids as in B was analyzed as in Figure 1D. The U1ΔSm gene was also used as a control. (E) Quantitation of RNA export inhibition by PHAXΔNES from the experiments like that shown in D.
	Figure 4. Effect of transcription from the CMV promoter on RNA export. (A) Diagram of the DNA constructs. Human β-globin cDNA fragments of various lengths or the U1ΔSm sequence were cloned between the CMV promoter and U1 terminator. The U1ΔSm gene was also used as a control. (B) Export of the transcripts produced from the microinjected plasmids harboring the cDNA fragments of various lengths (50–360 nt) or the U1ΔSm sequence between the CMV promoter and the U1 terminator was analyzed as in Figure 1B. The U1ΔSm gene was also used as a control. The bands indicated by the asterisk correspond to the 360 nt transcript whose 3′-end was properly formed. (C) Quantitation of RNA export inhibition by CTE WT from experiments like those shown in B. Since the β360 construct produced multiple bands for unknown reason, the bands near the expected size were quantified (B, asterisk). (D) Export of the transcripts produced from the microinjected plasmids harboring the cDNA fragments of 50 or 360 nt or the U1ΔSm sequence between the CMV promoter and the U1 terminator was analyzed as in Figure 1D. The U1ΔSm gene was also used as a control. The bands shown by the asterisk correspond to the 360 nt transcript whose 3′-end was properly formed. (E) Quantitation of RNA export inhibition by PHAXΔNES from the experiment shown in D. Since the β360 construct produced multiple bands, the bands near the expected size were quantified (D, asterisk).
	Figure 5. Effect of transcription from an mRNA transcription unit on RNA export. (A) Diagram of the DNA constructs. Human β-globin cDNA fragments of various lengths were cloned between the CMV promoter and the poly (A) signal from the bovine growth hormone (BGH) gene. The U1ΔSm gene was also used as a control. (B) Export of the transcripts produced from the microinjected plasmids harboring the cDNA fragments of various lengths (50–360 nt) between the CMV promoter and the BGH poly (A) signal was analyzed as in Figure 1B. Only the polyadenylated transcripts were visualized (see ‘Materials and Methods’ section). (C) The transcript from the plasmid harboring the U1ΔSm gene was analyzed as a control as in Figure 1B. (D) Quantitation of B and C.
	Figure 6. Effect of 3′-end processing signals on RNA export. Export of the transcripts produced from the plasmids harboring the β-globin 50-nt cDNA fragment between the CMV promoter and the U1 terminator (panel A) or between the U1 promoter and the BGH poly (A) signal (panel B), or from the plasmid harboring the U5 snRNA sequence between the U1 promoter and the BGH poly (A) signal (panel C) was analyzed as in Figure 5.
	Figure 7. Effect of poly (A) tail length on RNA export. (A) Diagram of the DNA construct as in Figure 6B is shown. (B) Effect of CTE and PHAXΔNES on export of the transcript from the plasmid was analyzed. For lanes 5–12, 0.8 pmol/oocyte of (dT)40 was coinjected with the plasmid. RNA length markers are shown on the left. The range of RNA length quantified in C is marked. (C) The RNA export inhibition by CTE observed in B, lanes 1–4, was calculated and plotted as a function of RNA length in the marked range in B. (D) One or two copies of a DNA fragment containing 70-nt poly (A) sequence were inserted into the XhoI site of the U1ΔSm gene. (E) Effect of CTE on export of the transcripts from the plasmids was analyzed. (F) Quantitation of RNA export inhibition by CTE from the experiments like that shown in E. (G) Various DNA constructs indicated in the table were microinjected into the nucleus of Xenopus oocytes. The nuclear fraction was prepared after 3 h, and immunoprecipitation was performed with either anti-PHAX antibody (α-PHAX) or anti-mouse IgG antibody (control). The co-precipitated RNA was recovered and analyzed by real-time RT-PCR. The efficiency of immunoprecipitation by each antibody is shown on the right.

References [+] :

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