"Roles of PSF protein and VL30 RNA in reversible gene regulation".

Xu Song, Ying Sun, and Alan Garen *

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

*To whom correspondence should be addressed.
Alan Garen, E-mail: alan.garen@yale.edu

Author contributions: X.S. and A.G. designed research; X.S. and Y.S. performed research; X.S. and A.G. analyzed data; and X.S. and A.G. wrote the paper.

Abbreviations: DBD, DNA-binding domain; OG, oncogenic gene; RBD, RNA-binding domain; EST, expressed sequence tag; pbt, PSF-binding tracts.

The mammalian protein PSF contains a DNA-binding domain (DBD) that coordinately represses multiple oncogenic genes in human cell lines, indicating a role for PSF as a human tumor-suppressor protein. PSF also contains two RNA-binding domains (RBD) that form a complex with a noncoding VL30 retroelement RNA, releasing PSF from a gene and reversing repression. Thus, the DBD and RBD in PSF are linked by a mechanism of reversible gene regulation involving a noncoding RNA. This mechanism also could apply to other regulatory proteins that contain both DBD and RBD. The mouse genome has multiple copies of VL30 retroelements that are developmentally regulated, and mouse cells contain VL30 RNAs that have normal and pathological roles in gene regulation. Human chromosome 11 has a VL30 retroelement, and a VL30 EST was identified in human blastocyst cells, indicating that the PSF-VL30 RNA regulatory mechanism also could function in human cells.

Cell Lines.

The human melanoma cell lines Th, yusac, and A2058, breast cancer cell line MCF7, adrenal tumor line NCI-H295R, cervical carcinoma cell line HeLa, pancreatic cancer cell line colo357, and the normal fibroblast line BJ were maintained as described in ref. 5. The MCF7-PSF line was cloned from MCF7 transfected with a PSF expression plasmid (provided by R. J. Urban, University of Texas Medical Branch, Galveston, TX). The MCF7-DBD line was cloned from MCF7 transfected with plasmid encoding a fragment of the PSF gene from positions 1 to 994.

Microarray Analysis.

RNA probes from MCF7 and MCF7-PSF cells were hybridized with the human HG-U133_Plus_2 Gene-Chip (Affymetrix, Santa Clara, CA) at the Yale W. M. Keck facility. The transcripts that showed at least a 4-fold increase or decrease of intensity in the MCF7-PSF cells relative to the MCF7 cells were analyzed by using NETAFFX, a web interface program from Affymetrix. Several of the microarray results were confirmed by semiquantitative RT-PCR assay.

Soft-Agar Colony Assay.

MCF7 or MCF7-PSF cells (3 X 10 ³ ) were suspended in 1 ml of 0.3% melted agar in MEM medium with
10% FBS and plated in 35-mm dishes containing a solidified layer of 0.5% agar in the same medium. After 3 weeks, 400 ml of PBS containing 0.5 mg of nitro blue tetrazolium was added to the dishes, and 1 day later, the colonies were scanned with an Epson Perfection 3200 scanner.

Semiquantitative RT-PCR.

To detect the effect of PSF on GAGE6 expression in tumor lines, the cells were transiently transfected with a control plasmid pCDNA3.1 or the plasmid encoding PSF by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h, semiquantitative RT-PCR was performed as described in ref. 5. GAPDH mRNA was used to normalize the amounts of RNA in the samples. To detect the inhibitory effect of VL30 RNA on the binding of PSF to GAGE6 promoter DNA, the cells were transfected with a plasmid containing the 318-bp VL30 cDNA fragment that contains the pbt sequences that bind to PSF protein. After 48 h, semiquantitative RT-PCR was performed to detect the expression level of GAGE6 and PSF transcripts. GAGE6 primers, 5'-GCCTCCTGAAGTGATTGGGCCTA-3' and 5'-CAGGCGTTTT CACCTCCTCTGGA-3'; PSF primers, 5'-ATGTCTCGGGATCGGTTCCGGA-3' and 5'-CCAACAAACAACCGACATCGCTG-
3'; and GAPDH primers, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'.

Electrophoretic Mobility Shift Assays (EMSA) and UV Cross-Linking.

Fifty nanograms of recombinant PSF protein, DBD of PSF, RBD of PSF, or the nuclear lysates containing 2 mg of total protein were mixed with 5 ng of ³² P-labeled RNA or DNA fragments. EMSA was performed as described in ref. 5.

Chromatin Immunoprecipitation (ChIP) Assay.

MCF7, MCF7-PSF, and MCF7-DBD cells were transfected with a plasmid encoding the 318-bp VL30 cDNA fragment, which contains the pbt sequences that bind to PSF protein. After 48 h, cells were cross-linked and processed for ChIP analyses by following the instruction of ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). The polyclonal antibody for PSF, provided by J. Patton (Vanderbilt University, Nashville, TN), was used to immunoprecipitate DNA. To analyze the PSF antibody-bound GAGE6 promoter DNA fragment, semiquantitative PCR was performed by using the following primers: GCCTTCTGCAAA-GAAGTCTTGCGC
and ATGCGAATTCGAGGCTGAG-GCAGACAAT.

Results:

PSF-Mediated Repression of OG and Inhibition of Oncogenesis.

The transcription patterns of the human breast tumor lines MCF7 that expresses a low level of PSF and MCF7-PSF that expresses a higher level of PSF, but otherwise are isogenic, were compared by microarray analyses (Fig. 1 A and B). The comparison identified 298 transcripts repressed by factors of 4–776 and 184
transcripts induced by factors of 4–147 in the MCF7-PSF line relative to the MCF7 line. Several of the microarray results were confirmed by semiquantitative RT-PCR. Most of the genes repressed by a factor of at least 32 (18 of 28) but none of the genes induced by a factor of at least 32 (0 of 14) have been

(A) Transcription of PSF. The MCF7 (low PSF) and MCF7-PSF (high PSF) cells were assayed for PSF transcription by semiquantitative RT-PCR.

(B) Microarray analysis. The transcription profiles of the MCF7 and MCF7-PSF cells were analyzed on Affymetrix microarray chips. Each bar in the figure indicates the number of transcripts repressed or induced in MCF7-PSF cells by a factor of 4–8, 8–16, 16–32, 32–64, and >64.

(A) Cell proliferation. Cells were grown in small flasks at 37°C as attached monolayers in MEM medium containing 10% FBS. Samples were recovered from three flasks every second day by treatment with trypsin, and the number of viable cells was counted. Each point is the average of three samples that agreed within +/- 5%.

(B) Colony formation. Single-cell suspensions were plated in soft agar, and the plates were incubated at 37°C for 19 days and photographed.

Each line was transiently transfected either with a control plasmid (lane 1) or with the same plasmid encoding PSF (lane 2). Semiquantitative RT-PCR assays were used to determine the transcription levels of GAGE6 and also of GAPDH to equalize the amount of mRNAin each pair. Cell lines: Th, A2058, and yusac are melanoma lines; MCF7 is a breast tumor line; H295R is an adrenal tumor line; and colo357 is a pancreatic tumor line.

characterized as OG (Table 1, which is published as supporting information on the PNAS web site). Because PSF is known to function only as a repressor, induction could result from repression of genes that encode repressors of the induced genes. MCF7-PSF cells proliferated at a slower rate and produced fewer
and smaller colonies in soft agar than MCF7 cells (Fig. 2 A and B), indicating that increased expression of PSF inhibits two basic properties of tumor cells, namely proliferation and colonization. PSF also represses the OG GAGE6 in several human tumor lines (Fig. 3). Thus, PSF appears to function as a human tumor-suppressor protein that coordinately represses transcription of multiple OG and inhibits oncogenesis.

Tumor suppressor genes are frequently mutated in tumor cells, inactivating or deleting the encoded tumor suppressor protein (6). Human PSF is located on 1p34, a region frequently associated with loss of heterozygosity in human tumors (7). Among 10 tumor lines tested for deletions of the PSF gene, we found one line with a deletion of the PSF gene, the cervical tumor line HeLa showed a short deletion of the DBD that did not extend either to the PSF promoter at the 5' end or the RBD at the 3' end (Fig. 4 A and B). Another mutation of the PSF gene, which is frequently detected in human papillary renal cell carcinomas, involves a translocation that fuses part of the PSF gene with the TFE3 gene, inactivating the normal function of the PSF protein (8, 9).

Mapping the Binding of PSF Protein to GAGE6 Promoter DNA.

The PSF-regulated gene GAGE6 was used as a model human OG for the remaining experiments. The GAGE6 regulatory DNA probably maps in the 2,241 bp region located between the 5' end of GAGE6-coding DNAand the 3' end of the closest flanking gene. Enzymatic digestion of this DNA segment generated five DNA fragments (Fig. 5A). Each fragment was ³² P-labeled, incubated with PSF protein, irradiated with UV, and analyzed by SDS/PAGE (Fig. 5B). The PSF protein bound only to the 262-bp

(A) Southern blot analysis. The genomic DNA isolated from HeLa (human cervical tumor) and BJ (human normal fibroblast) cell lines were hybridized with a ³² P-labeled PSF DNA probe spanning positions -32 to -154 of the PSF gene. The same amount of genomic DNA was added to each lane.

(B) RT-PCR analysis. DBD and RBD of PSF transcripts were amplified by RT-PCR.

(C) PCR analysis. Promoter region of PSF (-422 to -546) was amplified by PCR.

(A) Map of GAGE6 promoter fragments. The fragments were generated in the region -1 to -2241 flanking the 5' end of the GAGE6 coding region.

(B and C) Binding of PSF to GAGE6 promoter fragments. ³² P-labeled DNA fragments were mixed with PSF, irradiated with UV, and separated on 7.5% SDS/PAGE.

(B) Lanes: 1 (-629 to -1);2 (-1206 to -630); 3 (-1601 to -1207); 4 (-1979 to -1602); 5 (-2241 to -1980).

(C) Lanes: 1 (-2241 to -1980); 2 (-2180 to -1980); 3 (-2153 to -1980); 4 (-2109 to -1980); 5 (-2065 to -1980); 6 (-2021 to -1980).

(D) Sequence of the fragment from -2241 to -2181, containing the PSF-binding site.

Binding of PSF Protein, DBD, and RBD to GAGE6 Promoter DNA and hVL30 RNA in Vitro.

The DBD (region 1–994) and RBD (region 995-2124) domains were uncoupled on two PSF fragments, and
the fragments were tested for binding to GAGE6 promoter DNA and VL30 RNA in an EMSA. Binding to the DNA occurred with the DBD fragment but not with the RBD fragment (Fig. 6A), whereas binding to hVL30 RNA occurred with the RBD fragment but not with the DBD fragment (Fig. 6B). Addition of a VL30 RNA fragment containing the PSF-binding pbt tracts (5) inhibited binding of the PSF protein but not the DBD fragment to GAGE6 promoter DNA (Fig. 6C). These results demonstrate the separate roles of the DBD and RBD in the dual functions of PSF, as a DNA-binding tumor suppressor and a RNA-binding tumor promoter.

(A) Binding of PSF protein, DBD, and RBD to GAGE6 promoter DNA. ³² P-labeled GAGE6 DNA (-2241 to -1980), containing the PSF-binding site was mixed with intact PSF protein or a fragment containing the DBD or RBD, and the samples were analyzed by EMSA.

(B) Binding of PSF protein, DBD, and RBD to VL30 RNA. ³² P-labeled VL30 RNA was substituted for GAGE6 DNA, and the samples were analyzed as described in (A).

(C) Effect of VL30 RNA on binding of PSF protein and DBD to GAGE6 promoter DNA. Unlabeled VL30 RNA was added to samples containing ³² P-labeled GAGE6 DNA and mixed with PSF protein or DBD fragment. The molar ratio of VL30 RNA to GAGE6 promoter DNA was 0 in lanes 1 and 4, 10 in lanes 2 and 5, and 100 in lanes 3 and 6. The samples were analyzed by EMSA.

Binding of PSF Protein, DBD, and RBD to GAGE6 Promoter DNA and VL30 RNA in Vivo.

An anti-PSF antibody was used to immunoprecipitate PSF from human breast tumor cells expressing a low
level of PSF (MCF7 line), a high level of PSF (MCF7-PSF line), and a high level of the DBD fragment (MCF7-DBD line). The amount of GAGE6 promoter DNA coprecipitated with PSF was low in MCF7 cells and high in MCF7-PSF and MCF7-DBD cells. Transfection of VL30 cDNA encoding the pbt tracts resulted in a strong reduction of coprecipitated GAGE6 promoter DNA in MCF7 and MCF7-PSF cells and only a slight reduction in MCF-DBD cells (Fig. 7A). These results demonstrate that PSF protein and the DBD fragment bind to GAGE6 promoter DNA in vivo and that VL30 RNA binds to the RBD fragment, releasing PSF but not the DBD fragment from the DNA.

Effect of PSF Protein, DBD, and VL30 RNA on GAGE6 Transcription in Vivo.

GAGE6 is strongly transcribed in MCF7 cells and repressed in MCF7-PSF and MCF7-DBD cells (Fig. 7B). Transfection of

(A) Binding of PSF protein and DBDto GAGE6 promoter DNA in vivo and the effect of VL30 RNA. An anti-PSF antibody was used to immunoprecipitate PSF from human breast tumor lines expressing a low level of PSF (MCF7), a higher level of PSF (MCF7-PSF), or a high level of the DBD fragment (MCF7-DBD). The amount of GAGE6 promoter DNA coprecipitated with PSF was assayed by PCR. VL30 cDNA was transfected into cells in lanes 2, 4, and 6 but not in lanes 1, 3, and 5.

(B) Effect of PSF protein, DBD fragment, and VL30 RNA on GAGE6 transcription in vivo. The cell lines were the same as in A. Transcription of GAGE6, PSF, and VL30 was assayed by semiquantitative RT-PCR. VL30 cDNA was transfected into the cells analyzed in lanes 2, 4, and 6 but not in lanes 1, 3, and 5.

Effect of PSF Protein on Promoter Function.

To determine the effect of binding PSF to the CMV promoter that lacks a PSF-binding site, the 61-bp fragment from the GAGE6 promoter, which contains the PSF-binding site, was ligated to the 5' end of the CMV promoter. The CMV promoter with the PSF-binding site or the normal CMV promoter was ligated to the 5' end of a luciferase reporter gene that lacked a promoter. Each luciferase construct was transiently transfected into MCF7-PSF cells, and luciferase activity was assayed after 48 h. The luciferase activity was lower in the cells transfected with the hybrid CMV-luciferase construct than with the normal CMV-luciferase construct (Fig. 8), indicating that binding of PSF to a strong promoter represses transcription of the associated gene.

Discussion

Gene expression is a dynamic process involving selective regulation of gene transcription throughout the lifespan of an organism. We described earlier a novel mechanism for reversible gene transcription mediated by PSF protein and noncoding VL30 retroelement RNA (4, 5): PSF represses transcription of a gene by binding to the promoter, and VL30 RNA forms a complex with PSF that dissociates from the gene, activating transcription. Here, we show that the DBD and RBD of PSF can function independently: a PSF fragment containing only the DBD binds to a promoter and represses transcription but does not bind to VL30 RNA, whereas a PSF fragment containing only the RBD binds to VL30 RNA but not to a promoter (Figs. 6 and 7). Linking the DBD and RBD in the PSF molecule allows the RNA to reverse the binding of PSF to a promoter, probably by generating a conformational change through the RBD that

MCF7-PSF cells were transfected with a plasmid carrying a luciferase reporter gene either without a promoter (bar 1), with a wild-type CMV promoter (bar 2), or with a CMV promoter containing a PSF-binding site (bar 3). The values for luciferase activity are the means +/- SE for three experiments.

The mouse genome contains multiple copies of mVL30 retroelements that are developmentally regulated, and mouse cells contain mVL30 RNA that appear to have normal and pathological roles in gene regulation. A normal role for mVL30 RNA in mouse steroidogenesis is indicated by three findings, as follows. (i) PSF represses P450scc, the first gene in the steroidogenic pathway, by binding to the promoter region of the gene (2, 3). (ii) VL30 RNA reverses repression of P450scc by forming a complex with PSF protein (5). (iii) Pituitary-derived hormones concomitantly induce synthesis of mVL30 RNA and steroids in mouse steroidogenic cells (13). A plausible explanation for these findings is that the initial step in mouse steroidogenesis involves induced synthesis of mVL30 RNA by pituitary-derived hormones, followed by reversal of PSF-mediated repression of P450scc and activation of the steroidogenic pathway. In addition to a normal role for mVL30 RNA, there is evidence for a pathological role in oncogenesis: expression of mVL30 RNA is induced by various substances associated with oncogenesis and is elevated in transformed mouse cells (14–18). Human chromosome 11 contains a complete hVL30 retroelement with extensive sequence identity to mVL30 retroelements, and a 742-bp EST with extensive sequence identity to mVL30 RNA, including the pbt sequences that bind to PSF protein, was detected in human blastocyst cells (Data Set 1, which is published as supporting information on the PNAS web site), suggesting that hVL30 RNA has a role in human cells similar to the role of mVL30 RNA in mouse cells.

The association of retroelements such as VL30 with oncogenesis usually is attributed to cis effects on gene regulation and function, caused by insertional mutagenesis after reverse transcription of retroelement RNA. We showed that VL30 RNA also acts in trans by binding to PSF and reversing PSF-mediated gene repression. The ras family of oncogenic viruses contains VL30 sequences f lanking the ras coding region (19). Deletion of the VL30 segments strongly reduces the oncogenic potential of a ras virus (20), and infection by a ras virus induces synthesis of VL30 RNA in the infected cells (21). These findings support a transacting role for VL30 RNA in ras-mediated transformation, possibly involving reversal of PSF-mediated repression of OG.

Acknowledgments:

We thank Dr. James Patton for providing anti-PSF antibody and Dr. Randall Urban for providing the plasmid encoding human PSF. We thank Aiwei Sui, Ying Liu, and Henry Park for their technical assistance.
Partial support was provided by a gift account for Dr. Alan Garen’s laboratory.

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RNA was extracted from MCF7 (low PSF) and MCF7-PSF (high PSF) cells, and the biotinylated cRNA was hybridized with the human HG-U133_Plus_2 GeneChip (Affymetrix) as described in Methods. The transcripts that were repressed or induced in MCF7-PSF cells by a factor of >16 relative to the MCF7 cells are listed in Table 1:  http://www.pnas.org/cgi/content/full/0505179102/DC1#T1

Genes repressed or induced in MCF7-PSF cells by a factor of >16
 

	Oncogenic Genes		Other Genes
	Name (ref.)	Ratio	Name	Ratio	Total EST
Repressed by PSF	MB2 (1)	776	NJAC	64
	RAP1A (2)	724	BRAK	52
	IGFBP5 (3)	147	BF	52
	oral cancer candidate gene	69	LMP2	32
	CEACAM6 (4)	60	irx3	23
	S100A8 (5)	45	DNASE1	21
	S100A9 (5)	32	SLC22A4	18	23
	CXCL14 (6)	32	ZRP-1	17
	CP (7)	30	IFI35	16
	MUC16 (8)	30
	MDK (9)	24
	RTKN (10)	21
	ZNF91 (11)	21
	ANGPTL4 (12)	18
	BST2 (13)	18
	FVT1 (14)	18
	TCF-3 (15)	17
	GAGE6 (16)	16
Induced by PSF			CAV1	147
			COL12A1	69
			NR1I3	52
			GLRB	45
			ANXA1	39
			SCIN	34
			SLC7A11	32	7
			ORP3	28
			KRT13	24
			PLAC1	23
			NPy5R	17
			MGP	17
			GPNMB	16
			PBDX	16

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http://www.pnas.org/cgi/data/0505179102/DC1/1

A. Human VL30 DNA on chromosome 11.
B. Sequence of a human EST with extensive sequence identity to VL30 RNA.
 

A.

1 tgaagaatga aaaattactg gcctcttgtg agaacatgaa ctttttacct
51 cggagcccac cccctcccat ctgaaaaaca tttttgagat aaaggcctcc
101 tagaacaacc tcaaaatgaa ccgggtacat tgccaaatga taggacatga
151 ctccttagtt acgtagattc cttgatagga catgactcct tagttatgta
201 gattcctttg gcagaactcc ctagtgatgt aaacttgtat tttccctgcc
251 cagttctccc ccctttgagt tttactatat aagcctgtga aaaattttgg
301 ctgatggtcg agactcctct accctgtgca aaggtgtatg agtttcgacc
351 ccagagctct gtgtgctttc atgttgctgc tttatttcga ccccagagct
401 ctggtctgtg tgctttcatg ttgctacttt attaaatctt accttctaca
451 ttttatgtat ggtctcaatg tcttcttggg tacgtggctg tcccaggact
501 taagtgtctg agtaagggtc tcccttcggg ggtctttcat ttggtgcatt
551 ggccgggaat tcgagaatct ttcatttggt gcattggccg ggaaacagcg
601 cgaccaccca gaggtcctag acccacttag aggtaagatt ctttgttctg
651 ttttggtctg atgtcttgat gtctgtgttc tgatgtctgt gttctgtttc
701 taagtttggt gcgatcgcag tttcagtttt gcggacgctc agtgagaccg
751 cgctccgaga gggagtgcgg ggtggataag gatagacgtg tccaggtgtc
801 caccgtccgt tcaccctggg agacgtccca ggaggaacag gggaggatca
851 gggacgcctg gtggacccct ttgaaggcca agagaccatt tggtgttgcg
901 agatcgtggg ttcgagtccc acctcgtgcc cagttgcgag atcgtgggtt
951 cgagtcccac ctcgcgtttt gttgcgagat cgtgggttag agtcccacct
1001 cgcgccttgt tgcgagaccg tgggttcgag tcccacctcg cgtttggtca
1051 cgggatcgtg ggttcgagtc ccacctcgtg cagagggtct caattggccg
1101 gccttagaga ggccatctga ttcttctggt ttcttttttg tcttagtctc
1151 gtgtccgctc ttgttgtgac tactgttttt ctagaaatgg gacaatctgt
1201 gtccactccc ctttctctga ctctggagca ttggaaggag gtgcgggtca
1251 gagcccacaa ccagtcagtg gaggtcagaa agggtccgtg gcagaccttt
1301 tgcgcctccg agtggccaac gtttggagtg ggctggccac ctgagggtgc
1351 ttttgacttg tcactaatcg ccgccgtcag gcaaattgtt tttcaggagg
1401 aagggagtcg atgtgaaatc cgaccccgtc aggcttcttt ttctctgttg
1451 ctatcttgtt tttgtttgtg gtttttgtca tgagacacag acactgtacg
1501 tcggagataa agtgggatcc ctccctgtct gtatgtctgt gtgtctgtgt
1551 gtcttgttca cttgtgtgtc ctccattgga ccaccttctg attctgtttt
1601 gtttttggtt tgtctggttt caggtctggt cttggtccca aggagttgac
1651 tgattatctt ggtttggttt taggccggtc tcaggttctg ggaccttccc
1701 atggagacag gtccatttga gttggtcata gtgacagtaa gcttctctgg
1751 aaatcgcatg taagatgccc tgtattggtc ttgcttgtgt ttgtcatttc
1801 tgtggtactg tgattctata gtggttgcct aaaaaacggt ttctgtgtgt
1851 gtcttttgtg tctctttgtg ttcggacttg gactgatgac tgacgactgt
1901 ttttaagtta tgccttctga aataagccta aaattcctgt cagatcccta
1951 tgctgaccac ttcctttcag atcctcagct gccctgcctc ccactccaac
2001 tccagagagc agccagcggg tcacagtggt cccgcccatg gacctggagc
2051 ctagggaaaa atgagctcgg aaatccggag caaatgagga gtggtccctg
2101 agaagtcagt ggcctggatg ttgtggctgc tgaagaaaaa agaagaggaa
2151 gctgttcgag tagccggcca agagcgctgc aggttcccag gcagcttctc
2201 attcccctgt ccctcccatc ccgtctcttg ttaacagaaa aactgctttc
2251 actttaagat aagtgccggt tgcagccagc tgtgagagct gcactcccgt
2301 ctctgctcta aagttccctc ttctcagaag gtggcaccac cctgagctgc
2351 tggcagtgag tctgttccaa gttccagtga gggaggcatc cgcccacttg
2401 gggcttctgt ccaaggtaag gagcacctgt gagtctaact gccaggctct
2451 gatgggactc tcgtctctgt gggactaaaa agtcccaaca atctgaccaa
2501 ggtaacagga agttaagaca aagacagaga ccagagtcag aatcagagct
2551 gtgctgtgag acaaagagat aagagaagta aaatgctggt cacaaaagtc
2601 agggaaatta aaaaacttag atagtacctg gcaacaaaaa aaagcttttg
2651 gctgaagatc aatgtgttta agaaataaca aaaggggtgc taatacagaa
2701 gctgagtcct taaaagagtc cggtggccta cctgttaaag cagctagaaa
2751 aagagactgt gtttcatatt cctccaccga ccagtgcaaa acaagctaaa
2801 aagttcctgg gcactgcggg cttttgcaga ttgtggattc caggttttgc
2851 tgagttaaag agataaacag cccttcgtat agagaaaaaa taaagaaaac
2901 aatcttacat ggccttggat gctattaaga ctgccctaat gttatcccca
2951 gctatgggac tcctagatgt gactgagaac aaaggtattg ccaaaaaaag
3001 ttcttactca gagattggga ccctgaaaaa gacctgtggc atacttataa
3051 aaaattagac ctggtggctg taggatggcc tgcttgtctg cacatagtgg
3101 cttctggtca aggacgcaga taaattgact ctgagacaaa acttggcaca
3151 tgtcctaaaa agtgtggttc agcccccatg accgatggct gactaacgct
3201 cttgaaagca ttatccaact gttcccctga cggatggaca cattgtcaga
3251 gctttttgtt gactgaacga gtgaccttcg ctccccctgc tatcctcaat
3301 ctcactactg cctgagactt cacctactca tcattgtgct gacattctgg
3351 cagaaggaac tcatactcga aatgatctaa aggatcagat cagccttggc
3401 ctgagagttt gagctggtac acggatggca gtagcctgga ggttgaaggt
3451 aagcggaagg cggggacagc agtgcagtgg tggacagaaa gcaagtgatc
3501 taggccagca gcctccttaa agggacctca gcccagaaag ctaaacttgt
3551 ggctttaata caagctctat aaatggtaaa agaaaagtct acacggacag
3601 caggtatgct cttgccactg tacagagcaa tatacagaca aagagagctg
3651 ttgacatcag cagagaaaga cctaagatgc tgtggctaaa aaaatcaggt
3701 gacaaatcta accgcccagg catcctaaag agcaatgatc ctgacagtct
3751 gaagactatc aagttataga caaattaaga ctggtaaaag aaaccctgta
3801 taaaatagta gaaactgaag aaaaaaaaac tagtcctctc atgagaagac
3851 agacctgaca tctactgaaa aatagacttt actgaaaaaa atatgtgtat
3901 aaataccttc tggtttttgt gaacgttctc aagatggata aaagcttttc
3951 cttataaaac tgagactgct cagatagtca tcaagaagat tattaaaaat
4001 tttccaaggt tcggagtgcc aaaagcaata gtgtcagata atggtcctgt
4051 ccttgttgcc caggtaagtc agggtgtggc caagtattta gaggtcaaat
4101 aaaaattgta ttatgtgtac agacctcaga actcaggaca gatagaaata
4151 ataaataaaa ctctaaacag accttgacaa aattaatcct agagactggc
4201 acagacttac ttggtactcc ttccccttgc cctatttaga actgagaata
4251 ctccctcttg attcggtttt actatttttg agatccttta tggggctcct
4301 aggcccatca ctgtcttaaa taatgtgttt aaacctatgt tgttataata
4351 atgatctgta tactaagtta taaggcttac aggtggtgca gaaagaagtc
4401 tggtcacaac tggctacagt gaacaagctg ggtaccccaa ggacatctta
4451 ccagttccag ccagagatct gatctgcgta cacctgcgtc atgctgagac
4501 cctcaagcct cactagaagg gtccctgcct agttctgttt actaatccgc
4551 cttattctgt ttttgttccc atgttaaaga tagagtaaat acagtattct
4601 ccacatagag atatagactt ctgaaattct aagattagaa ttacttacaa
4651 gaagaagtgg ggaatgaaga atgaaaaatt actggcctct tgtgagaaca
4701 tgaacttttt acctcggagc ccaccccctc ccatctgaaa aacatttttg
4751 agataaaggc ctcctagaac aacctcaaaa tgaaccgggt acattgccaa
4801 atgataggac atgactcctt agttacgtag attccttgat aggacatgac
4851 tccttagtta tgtagattcc tttggcagaa ctccctagtg atgtaaactt
4901 gtattttccc tgcccagttc tccccccttt gagttttact atataagcct
4951 gtgaaaaatt ttggctgatg gtcgagactc ctctaccctg tgcaaaggtg
5001 tatgagtttc gaccccagag ctctgtgtgc tttcatgttg ctgctttatt
5051 tcgaccccag agctctggtc tgtgtgcttt catgttgcta ctttattaaa
5101 tcttaccttc tacattttat gtatggtctc agtgtcttct tgggtacgtg
5151 gctgtcccgg gacttaagtg tctgagtaat ggtctccctt cgggggtctt
5201 tcatttggtg cattggccag gaattcgaga atctttca

B.

1 ccttagagag gccatctgat tcttctggtt tcttttttgt cttagtctcg
51 tgtccgctct tgttgtgact actgtttttc tagaaatggg acaatctgtg
101 ttcactcccc tttctctgac tctggagcat tggaaggagg tgcgggtcag
151 agcccacaac cagtcggtgg aggtcagaaa gggtccgtgg cagacctttt
201 gcgcctccga gtggccaacg tttggagtag gctggccacc taagggtgct
251 tttgacttgt cactaatcgc caccatcagg caaattgttt ttcaggagga
301 agggagtcga tgtggaatct gcccccgtca ggaaatagtg tttggcttgg
351 ctggttgtac aagtccaggc gtggacgagt gtgcttagag atactggtgt
401 cttctttttc tctgttgcta tcttgttttt gtttgtggtt tacggttttt
451 gtgtgtgtct ttttttttgt ctctttgtgt tcggacttgg actgatgact
501 gacgactgtt tttaagttat gccttctgaa ataagcctaa aaatcctgtc
551 agatccctat gctgaccact tcctttcaga tcaacagctg ccctgcctcc
601 cactccaact ccagagagca gccagcgggt cacagtggtc ccgtccatgg
651 atacagccag ctgtgagagc tgcactccct tccatgcccc acgtgttttc
701 tcgtctcagg cgacccctct ttgagctgct gacagtgagt ct

CORRECTION:

MEDICAL SCIENCES. For the article "Roles of PSF protein and VL30 RNA in reversible gene regulation," by Xu Song, Ying Sun, and Alan Garen, which appeared in issue 34, August 23, 2005, of Proc. Natl. Acad. Sci. USA (102, 12189-12193; first published August 3, 2005; 10.1073/pnas.0505179102) and was discussed in "Normal and pathological functions of mammalian retroelements," a commentary by Albert Deisseroth in issue 35, August 30, 2005 (102, 12292-12293; first published August 23, 2005; 10.1073/pnas.0505866102), the authors note that the human genome contains a sequence on chromosome 11 (clone RP11-419K3) almost identical to a mouse retroelement VL30. This sequence was registered in GenBank as AC019351 on September 9, 2000, but was removed from the database after publication of this article because it is now considered to be a contaminating mouse sequence. The authors also referenced a 742-bp EST sequence (CX757918 [GenBank] ) that has extensive sequence identity to a region of a mouse VL30 RNA. The EST was cloned from a pleuripotent cell line derived from a human blastocyst inner cell mass, but the apparent lack of a coding sequence in the human genome suggests that the EST could be a mouse contaminant. The only conclusion in the papers that has changed concerns the presence of a VL30 gene in the human genome. All other conclusions remain valid.

This new study by Xu Song, Ying Sun, and Alan Garen reveals that a noncoding RNA,  found in the mouse genome, is capable of de-repressing the activity of a specific protein repressor, resulting in a more than 16-fold activation of 14 human genes. The mouse gene coding for this RNA has been isolated and sequenced as a 5238 bp sequence, and an expression sequence tag has been isolated and sequenced as a 742 bp sequence. The RNA molecule is believed to be involved in steroidogenesis, oncogenesis, and perhaps the promotion of established neoplasms.

1. Hovsepian JA, and Frenster JH, "Sense and antisense during RNA initiation of the DNA transcription bubble".

2. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter".

3. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells".

4. Ostertag EM,  and Kazazian HH, "Genetics:  LINEs in mind".

5. Muotri AR, Chu VT, Marchetto MCN, Deng W, Moran JV, and Gage FH, "Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition".

6. Deisseroth A, "Normal and pathological functions of mammalian retroelements".

7. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin".

8. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".
 

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A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).