(A) The potential Sm binding site elements ofC. differed somewhat from those of additional U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur individually of Sm proteins. == Summary == Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II experienced a TMG cap, while those expected to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs. == Background == The 2 2,2,7-trimethylguanosine TM5441 (TMG) cap structure was first found at the 5′-end of low-molecular-weight nuclear RNAs, such as U1 small nuclear RNAs (snRNA), U2 snRNA, U5 snRNA and U3 small nucleolar RNAs (snoRNAs) fromNovikoffhepatoma cells (examined in [1]). In nematodes and some additional metazoans, 5′-end TMG caps have been extensively characterized fortrans-spliced mRNAs and practical non-coding RNAs (ncRNAs), such as U1, U2, U4 and U5 snRNAs, and for some of the snoRNAs and spliced innovator (SL) RNAs [2]. All TMG-capped snRNAs are transcribed by RNA polymerase II. In mammals, hypermethylation of the m7G cap of the U1, U2, U4, and U5 snRNAs to a TMG cap happens in the cytoplasm, and depends on some of the Sm core proteins [3,4]. Unlike the assembly of the spliceosomal snRNPs, which take place in the cytoplasm, biogenesis of the vertebrate snoRNPs takes place in the nucleolar compartment, and hypermethylation of the TMG-capped snoRNAs is not associated with Sm proteins [5-7]. In candida, U1, U2, U4, and U5 snRNAs also have a TMG cap structure [8] and hypermethylation of the cap structure TM5441 is dependent within the Sm proteins [9,10], and a conserved methyltransferase, which is essential for hypermethylation of the m7G caps of snRNAs and snoRNAs[11]. Although the cellular function of the TMG cap is not obvious, it is believed the trimethylguanosine caps are necessary for the snRNAs to fulfill their cellular functions [12]. The TMG cap is an important component of the nuclear localization transmission of U snRNPs, and there is evidence that core U-snRNPs without a TMG structure cannot be imported into the nucleus [13,14]. Biochemical dedication of a TMG cap structure offers traditionally required several experimental methods [15], and antibodies that identify specific constructions offers consequently greatly facilitated screening for ncRNAs with TMG caps. Capped ncRNAs have been purified from total cellular RNA of vegetative microplasmodia by preparative immunoprecipitation with anti-TMG antibodies [16,17]. These antibodies specifically react with TMG-capped RNAs such as U1, U2 and U4 snRNAs. This can also be used to isolate snRNPs in one step from nuclear components of eukaryotic cells by affinity chromatography on a preparative level [18]. Several strategies have been endeavored to detect or discover novel ncRNAs, including both experimental and computational screening [19-22]. Results from the recent tiling microarray studies also show that far larger portions of the eukaryote transcriptome than formerly believed are actually transcribed in the form of non-coding transcripts [23]. Systemic examination of the 5′-end TMG cap structures in the novel ncRNAs would be useful contribution towards decoding the cellular functions of these transcripts. Here we combined immunoprecipitation and an ncRNA microarray [24] to identify 5′-end TMG cap structures CDKN1B in most of the presently characterized ncRNAs inC. elegans[19], and analyzed important characters connected TMG-capped ncRNAs, such as function, biogenesis and expression. == Results == == ncRNAs precipitated from the anti-TMG antibodies == We 1st filtered TM5441 and immunoprecipitated worm total RNA with the K121 anti-TMG antibody. However, after having been made aware that this antibody may have a reduced specificity for TMG caps and has shown some cross-reactivity with mono-methylated cap constructions [25,26], we repeated all subsequent experiments with the R1131 antibody [17]. RNAs in both precipitate and supernatant acquired with both antibodies were extracted and reversely transcribed. The cDNA from precipitate and supernatant was labeled with Cy3 or Cy5, respectively, and hybridized to a microarray with probes for 127 ncRNAs [24]..