SINEs as Potential Expression Cassettes: Impact of Deletions and Insertions on Polyadenylation and Lifetime of B2 and Ves SINE Transcripts Generated by RNA Polymerase III.

Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called ß- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are ß- and τ-signals. Extended regions outside of ß- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5'-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of ß- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between ß- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications.

Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

Tail Wags Dog's SINE: Retropositional Mechanisms of Can SINE Depend on Its A-Tail Structure.

SINEs, non-autonomous short retrotransposons, are widespread in mammalian genomes. Their transcripts are generated by RNA polymerase III (pol III). Transcripts of certain SINEs can be polyadenylated, which requires polyadenylation and pol III termination signals in their sequences. Our sequence analysis divided Can SINEs in canids into four subfamilies, older a1 and a2 and younger b1 and b2. Can_b2 and to a lesser extent Can_b1 remained retrotranspositionally active, while the amplification of Can_a1 and Can_a2 ceased long ago. An extraordinarily high Can amplification was revealed in different dog breeds. Functional polyadenylation signals were analyzed in Can subfamilies, particularly in fractions of recently amplified, i.e., active copies. The transcription of various Can constructs transfected into HeLa cells proposed AATAAA and (TC)n as functional polyadenylation signals. Our analysis indicates that older Can subfamilies (a1, a2, and b1) with an active transcription terminator were amplified by the T+ mechanism (with polyadenylation of pol III transcripts). In the currently active Can_b2 subfamily, the amplification mechanisms with (T+) and without the polyadenylation of pol III transcripts (T-) irregularly alternate. The active transcription terminator tends to shorten, which renders it nonfunctional and favors a switch to the T- retrotransposition. The activity of a truncated terminator is occasionally restored by its elongation, which rehabilitates the T+ retrotransposition for a particular SINE copy.

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators.

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3'-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Identification of nucleotide sequences and some proteins involved in polyadenylation of RNA transcribed by Pol III from SINEs.

We have previously reported that not only transcripts of RNA polymerase II (pol II), but also one type of RNA transcribed by RNA polymerase III (pol III), undergo AAUAAA-dependent polyadenylation. Such an unusual feature is inherent in Short Interspersed Elements (SINEs) from genomes of certain mammals. For polyadenylation of its transcript, SINE should contain, besides an AATAAA hexamer and a transcription terminator, two specific regions: ß, located downstream of box B of a promoter, and τ, preceding AATAAA. Here, using nucleotide substitutions in SINEs B2 (mouse) and Ves (bat), we identified nucleotides of ß regions necessary for polyadenylation of their transcripts. These sequences (ß signals) are the following: ACCACATgg in B2 and GGGCATGT in Ves. Using this approach, we identified τ signal of SINE B2 (GCTACagTGTACTTACAT), where TGTA tetramer is most important for polyadenylation. In Ves, τ region is a long polypyrimidine motif which is able to interact with PTB protein in Ves transcripts. We demonstrated by knockdown that B2 and Ves transcript polyadenylation is performed by canonical poly(A) polymerase with the participation of proteins CSPF-160 and Fip1, the known factors of mRNA polyadenylation. We also showed that a factor CFIm partaking in polyadenylation of many mRNAs, is involved only in polyadenylation of B2 transcripts. CFIm seems to interact with τ signal of В2 RNA and thereby facilitates the recruiting of other proteins engaged in polyadenylation. Thus, SINEs utilize at least some proteins involved in polyadenylation of pol II transcripts to polyadenylate their pol III transcripts.

Polyadenylation of RNA transcribed from mammalian SINEs by RNA polymerase III: Complex requirements for nucleotide sequences.

It is generally accepted that only transcripts synthesized by RNA polymerase II (e.g., mRNA) were subject to AAUAAA-dependent polyadenylation. However, we previously showed that RNA transcribed by RNA polymerase III (pol III) from mouse B2 SINE could be polyadenylated in an AAUAAA-dependent manner. Many species of mammalian SINEs end with the pol III transcriptional terminator (TTTTT) and contain hexamers AATAAA in their A-rich tail. Such SINEs were united into Class T(+), whereas SINEs lacking the terminator and AATAAA sequences were classified as T(-). Here we studied the structural features of SINE pol III transcripts that are necessary for their polyadenylation. Eight and six SINE families from classes T(+) and T(-), respectively, were analyzed. The replacement of AATAAA with AACAAA in T(+) SINEs abolished the RNA polyadenylation. Interestingly, insertion of the polyadenylation signal (AATAAA) and pol III transcription terminator in T(-) SINEs did not result in polyadenylation. The detailed analysis of three T(+) SINEs (B2, DIP, and VES) revealed areas important for the polyadenylation of their pol III transcripts: the polyadenylation signal and terminator in A-rich tail, ß region positioned immediately downstream of the box B of pol III promoter, and τ region located upstream of the tail. In DIP and VES (but not in B2), the τ region is a polypyrimidine motif which is also characteristic of many other T(+) SINEs. Most likely, SINEs of different mammals acquired these structural features independently as a result of parallel evolution.

Short interspersed elements (SINEs) of squamate reptiles (Squam1 and Squam2): structure and phylogenetic significance.

Short interspersed elements (SINEs) are important nuclear molecular markers of the evolution of many eukaryotes. However, the SINEs of squamate reptile genomes have been little studied. We first identified two families of SINEs, termed Squam1 and Squam2, in the DNA of meadow lizard Darevskia praticola (Lacertidae) by performing DNA hybridization and PCR. Later, the same families of retrotransposons were found using the same methods in members of another 25 lizard families (from Iguania, Scincomorpha, Gekkota, Varanoidea, and Diploglossa infraorders) and two snake families, but their abundances in these taxa varied greatly. Both SINEs were Squamata-specific and were absent from mammals, birds, crocodiles, turtles, amphibians, and fish. Squam1 possessed some characteristics common to tRNA-related SINEs from fish and mammals, while Squam2 belonged to the tRNA(Ala) group of SINEs and had a more unusual and divergent structure. Squam2-related sequences were found in several unannotated GenBank sequences of squamate reptiles. Squam1 abundance in the Polychrotidae, Agamidae, Leiolepididae, Chamaeleonidae, Scincidae, Lacertidae, Gekkonidae, Varanidae, Helodermatidae, and two snake families were 10(2) -10(4) times higher than those in other taxa (Corytophanidae, Iguanidae, Anguidae, Cordylidae, Gerrhosauridae, Pygopodidae, and Eublepharidae). A less dramatic degree of copy number variation was observed for Squam2 in different taxa. Several Squam1 copies from Lacertidae, Chamaeleonidae, Gekkonidae, Varanidae, and Colubridae were sequenced and found to have evident orthologous features, as well as taxa-specific autapomorphies. Squam1 from Lacertidae and Chamaeleonidae could be divided into several subgroups based on sequence differences. Possible applications of these SINEs as Squamata phylogeny markers are discussed.

Transcripts synthesized by RNA polymerase III can be polyadenylated in an AAUAAA-dependent manner.

It is well known that nearly all eukaryotic mRNAs contain a 3' poly(A) tail. A polyadenylation signal (AAUAAA) nearby the 3' end of pre-mRNA is required for poly(A) synthesis. The protein complex involved in the pre-mRNA polyadenylation is coupled with RNA polymerase II during the transcription of a gene. According to the commonly accepted view, only RNAs synthesized by RNA polymerase II can be polyadenylated in an AAUAAA-dependent manner. Here we report the polyadenylation of short interspersed elements (SINEs) B2 and VES transcripts generated by RNA polymerase III. HeLa cells were transfected with SINE constructs with or without polyadenylation signals. The analyses of the SINE transcripts showed that only the RNAs with the AAUAAA-signal contained poly(A) tails. Polyadenylated B2 RNA was found to be much more stable in cells than B2 RNA without a poly(A) tail.

PCR-based approach to SINE isolation: simple and complex SINEs.

Highly repeated copies of short interspersed elements (SINEs) occur in eukaryotic genomes. The distribution of each SINE family is usually restricted to some genera, families, or orders. SINEs have an RNA polymerase III internal promoter, which is composed of boxes A and B. Here we propose a method for isolation of novel SINE families based on genomic DNA PCR with oligonucleotide identical to box A as a primer. Cloning of the size-heterogeneous PCR-products and sequencing of their terminal regions allow determination of SINE structure. Using this approach, two novel SINE families, Rhin-1 and Das-1, from the genomes of great horseshoe bat (Rhinolophus ferrumequinum) and nine-banded armadillo (Dasypus novemcinctus), respectively, were isolated and studied. The distribution of Rhin-1 is restricted to two of six bat families tested. Copies of this SINE are characterized by frequent internal insertions and significant length (200-270 bp). Das-1 being only 90 bp in length is one of the shortest SINEs known. Most of Das-1 nucleotide sequences demonstrate significant similarity to alanine tRNA which appears to be an evolutionary progenitor of this SINE. Together with three other known SINEs (ID, Vic-1, and CYN), Das-1 constitutes a group of simple SINEs. Interestingly, three SINE families of this group are alanine tRNA-derived. Most probably, this tRNA gave rise to short and simple but successful SINEs several times during mammalian evolution.

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule.

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.