Gravina P et al. (2002), Complementary DNA analysis, expression and subc...

XB-ART-7009

Gene 2002 May 15;2901-2:193-201. doi: 10.1016/s0378-1119(02)00561-9.

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Complementary DNA analysis, expression and subcellular localization of hnRNP E2 gene in Xenopus laevis.

Gravina P , Campioni N , Loreni F , Pierandrei-Amaldi P , Cardinali B .

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The cloning and sequencing of complementary DNAs corresponding to the two copies (a and b) of the Xenopus laevis gene for hnRNP E2 is presented. Comparison of the two sequences reveals that while they are somewhat divergent at the nucleotide level, they are very conserved at the amino acid level. The analysis also showed two transcripts of different length (alpha and beta), likely generated by alternative processing. There are indications that either gene copy can generate both type of transcripts. Northern blot analysis in oocytes and developing embryos showed that hnRNP E2 RNA is constantly present and that increases in amount at tadpole stage. A semiquantitative reverse transcriptase polymerase chain reaction analysis performed with RNA from developing embryos showed that long (alpha) transcript accumulation is constant during development, whereas the short one (beta) accumulation increases at later stages, thus determining the observed increase in total RNA. Nucleo-cytoplasm localization experiments indicated that in oocyte hnRNP E2 is exclusively cytoplasmic, whereas in somatic cells it is distributed in both compartments. Comparison of the amino acid sequence of the two X. laevis hnRNP E2 with the corresponding mammalian sequences shows a high homology along the molecule except for the region subjected to alternative splicing, which is completely different. Moreover, there are indications that the homologous of mammalian hnRNP E1 gene, very related to and derived from hnRNP E2 by retrotransposition, is not expressed or even not present in X. laevis, suggesting that mammalian hnRNP E1 gene may have originated after mammal/amphybia divergence.

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Species referenced: Xenopus laevis
Genes referenced: hnrnpc hnrnpdl myc pcbp3

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	Fig. 1. Nucleotide sequence of the coding region of the X. laevis hnRNP E2 a cDNA (AJ243591), clone 2.2, and deduced amino acid sequence, and comparison with the hnRNP E2 b copy (BN000061), covered by clone 11.3 (nt 205â1062) and by part of the EST sequence BG160589 (nt 1â204). The initiation codon is boxed. The nt and aa that differ in the two copies are underlined. The stars indicate nucleotide omission in copy b. KH domains are marked by shaded boxes, for simplicity, only in copy a. The bars indicate theDNAfragments used to prepare oligos for the differential analysis of the two alternative transcripts shown in Fig. 3 and described in Section 2. Marks on the sequence and nt and aa numeration of hnRNP E2 a are also considered for hnRNP E2 b. The first part (nt 1â204) of the hnRNP E2 b X. laevis cDNA sequence was taken from the Gene Bank, n. BG160589 (Clifton et al., 2001a), the remaining part was isolated by the authors.
	Fig. 2. Alignment of the amino acid sequence of X. laevis hnRNP E2 with human and mouse proteins hnRNP E2 and hnRNP E1. Genbank accession numbers: human hnRNP E2, X78136 (Leffers et al., 1995), mouse mCBP-2, X75947 (Goller et al., 1994), mouse PCBP-2 variant E, X97982 (Funke et al., 1996), mouse variant hnRNP X, L19661 (Hahm et al., 1993), human hnRNP E1, X78137 (Leffers et al., 1995), mouse PCBP-1, AF139894 (Makeyev et al., 1999). The two X. laevis alternative forms in the region 194â227 are here called a and b, regardless their different gene origin. KH domains are shaded. The stars indicate aa omissions. Numeration starts from the first methionine of the hnRNP E2 a X. laevis transcript (see Fig. 1) to which all the other sequences are referred.
	Fig. 3. Expression of X. laevis hnRNP E2 and abundance of the a and b transcripts during development. (A) 2 mg of poly(A) RNA from oocytes and embryos were analyzed on a Northern gel and hybridized with X. laevis hnRNP E2 and ribosomal protein L4 probes. (B) Southern blot analysis of RT-PCR products of total RNA from embryos of different stages obtained using the oligos indicated in Fig. 1 as described in Material and methods. PCR reactions were loaded on a 3.5% agarose gel, blotted and hybridized to a 50 labeled oligo which recognizes both a and b transcripts. The size of the two fragments on the gel matches the predicted sizes of the amplified regions, 248 nt for the a form and 146 for the b form. Molecular weight markers are indicated on the left.
	Fig. 4. Utilization of hnRNP E2 mRNA in oocyte and embryos. (A) Cytoplasmic extracts from oocytes and embryos of different stages fractionated on sucrose gradients. Poly(A) RNA was extracted from polysome and subpolysomal fractions and analyzed by Northern blot hybridization with the hnRNP E2 probe. The RNA loaded on each gel corresponds to the same oocyte and embryo number of individuals. As a control, the same filter was hybridized to a probe for histone H3. Stages are indicated at left. (B) Analysis of hnRNP E2 in oocytes and embryos by Western blot. A total of 90 mg of protein from cytoplasmic soluble extracts were loaded on protein gels, blotted and incubated with an antiserum versus X. laevis hnRNP E2. The filter was also incubated with an antiserum developed versus Xenopus recombinant La. Numbers refer to developmental stages.
	Fig. 5. Nucleo-cytoplasm partition of hnRNP E2. (A) Extracts from manually dissected oocyte nuclei and cytoplasms (the equivalent of four oocytes) were analyzed by Western blot with an hnRNP E2 antiserum. Antibodies were stripped from the filter which was then incubated with an antiserum versus the X. laevis La antigen. (B) Cultured A6 cells were processed for immunofluorescence as indicated in Material and methods, using X. laevis hnRNP E2 affinity purified antibodies (top). Cells were transiently transfected with the plasmid pCMV hnRNP E2 that encodes for myc-tagged hnRNP E2 (bottom). Immunostaining was performed using the mAB 9E10, that recognizes the myc region of the protein. The arrow points to a nontransfected cell.
	Fig. 6. Binding of XhnRNP E2 to L4 mRNA. In vitro-transcribed radioactive L4 mRNA (1 pmole) was incubated with 0.1 mg of recombinant X. laevis GST-hnRNPE2 or La protein alone or in the presence of 7.5 mg of a 0.25HS fraction. After UV crosslinking and RNAse digestion the samples were analyzed on a 13% polyacrylamide/SDS gel. Molecular mass markers are indicated.