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for cDNA synthesis, you're mostly measuring rRNA.

Several hundred ribosome assembly factors have been identified in budding yeast and, more recently, human cells (; ; ). The field is now facing the daunting task of assigning precise functions to them, identifying their binding sites or substrates on precursor ribosomes, and establishing the timing of their intervention—that is, a precise sequence of events. We show here that DIMT1L is involved in earlier processing steps than WBSCR22-TRM112 ( and and Supplemental Figure S3). This is consistent with the respective subcellular localizations of these proteins: the nucleolus and nucleoplasm. The nuclear localization of WBSCR22 is consistent with previous reports (; ), but these studies failed to detect it in perinuclear vesicles (see later discussion).

Cells lacking DIMT1L accumulate the 43S, 26S, and 21S/21S-C pre-rRNAs. Hence the cleavages occurring at sites A0 and 1, which are normally concomitant, are disconnected in these cells, and cleavage at sites 1, C, and E is inhibited. Cells lacking WBSCR22 or TRMT112 accumulate the 18S-E precursor. This indicates inhibition of cleavage at site 3, a processing step reported to occur in the cytoplasm (). A likely explanation of this processing phenotype is that in the absence of WBSCR22-TRMT112, preribosomes are simply not exported to the cytoplasm. In agreement with this hypothesis, investigators have observed retention of fluorescently labeled pre-40S in the nucleus upon WBSCR22 depletion (), and the 3′-extended forms of 18S-E that accumulates under these conditions are reported to be nuclear ().

qRT‐PCR; housekeeping gene; 28S rRNA; HeLa cells; insect cells; baculovirus; adenovirus

18S_rRNA cDNA ORF clone, Aspergillus niger CBS 513.88

When expression of a recombinant construct was not induced (lanes without tetracycline addition), cells depleted of endogenous DIMT1L accumulated the 26S pre-rRNA and/or the long truncated (asterisk) 18S precursor fragment (, lanes 3, 4, 7, and 11). This is consistent with our earlier results ( and Supplemental Figure S3). When synthesis of the DIMT1L-Y131G variant was induced by addition of tetracycline to the medium, accumulation of these diagnostic RNAs was suppressed (, compare lanes 11 and 12 and see quantitation). Hence the catalytic activity of DIMT1L is not required for pre-rRNA processing. As control, expression of the wild-type construct with the siRNA resistant mutation also suppressed the processing defect (disappearance of the 18S-E truncated form [asterisk] in , lane 8, as compared with lane 7), indicating efficient rescue.

Similarly, 0.2 μg/ml tetracycline proved optimal for production of the recombinant forms of WBSCR22, in most cases yielding levels similar to that of the endogenous protein (, lanes 6, 10, 14 and 18). Synthesis of endogenous WBSCR22 was efficiently suppressed by siRNAs (, lanes 3/4, 7/8, 11/12, 15/16, and 19/20). In contrast, the recombinant constructs were stably expressed, indicating that, as expected, the mRNAs encoding them were siRNA resistant (, lanes 8, 12, 16, and 20). In the absence of expression of a complementing construct, cells depleted of endogenous WBSCR22 accumulated the 18S-E pre-rRNA intermediate and the long and short truncated 18S precursor fragments (single and double asterisks; , lanes 3, 4, 7, 11, 15, and 19). This is consistent with our earlier results ( and Supplemental Figure S3). Accumulation of these fragments was suppressed to a similar extent in cells producing the wild-type protein (, lane 8) or any of the three catalytically defective variants (, lanes 12, 16, and 20). This demonstrates that the catalytic activity of WBSCR22, like that of DIMT1L, is not required for pre-rRNA processing in human cells.

18S_rRNA available at GenScript with gene synthesis service.

In budding yeast, pre-rRNA processing requires the presence of the methyltransferases Dim1 and Bud23 within preribosomes but not their RNA-modifying catalytic activity (; ; ; Introduction).

The ribosome is an extraordinary complex nanomachine whose blueprint has been extremely conserved throughout evolution across all three kingdoms of life (). This is strikingly illustrated when one compares the ribosomes of simple and more complex eukaryotic cells, such as budding yeast and human cells (; ). This resemblance has led to the assumption that ribosome biogenesis must be quite similar in yeast and humans. Recent research indicates that this is true, but only to some extent, as important specificities have begun to emerge (; ). For example, the RNA exosome involved in ITS2 processing in yeast is additionally required for ITS1 maturation in humans, and the endoRNase MRP, which cleaves ITS1 in yeast, does not seem to play such a role in humans (; ). Such specificities in pre-rRNA processing are making it absolutely imperative to conduct research directly on human cells. If ribosomopathies—that is, human syndromes associated with ribosome assembly dysfunctions ()—were to be modeled only in budding yeast, potential therapeutic targets might be selected for the wrong reasons and others left totally unnoticed.

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iScript cDNA Synthesis Kit | Life Science Research | Bio …

comprised by the 28S and 18S rRNA ..

In yeast cells, depletion of the 40S subunit assembly factor Pno1 (also known as Dim2) strongly inhibits Dim1-mediated dimethylation (; Introduction). In pre-40S ribosomes, Dim1 and Pno1/Dim2 are located to either side of the incipient platform (). This physical proximity likely underlies the functional interaction between Dim1 and Dim2. The position of the platform (“Pt”) on mature 40S is shown in . Using the primer extension assay described, we tested whether efficient 18S rRNA dimethylation in human cells might likewise require PNO1, the human homologue of Dim2, and we found that it does (). It is not yet clear why PNO1 depletion consistently affected 18S rRNA dimethylation more than DIMT1L depletion. We hypothesized that this is because in cells depleted for PNO1, the substrate fails to adopt a suitable conformation to be modified. Finally, we were interested to know whether PNO1 is required for the metabolic stability of DIMT1L, and we found, in cells efficiently depleted for PNO1, that it is not ().

Amplifluor® Human/Mouse 18S rRNA Primer Set (JOE labeled) ..

In conclusion, our primer extension mapping data demonstrate that DIMT1L and WBSCR22-TRMT112 are responsible, respectively, for and m7G1639 in human 18S rRNA. We further show that DIMT1L-mediated dimethylation in human resembles dimethylation in yeast in that it requires hDIM2 (PNO1), but differs from yeast dimethylation in that it occurs in the nucleus before preribosome export to the cytoplasm.

primer for 1st strand cDNA synthesis…

Primer extension mapping was performed on total RNA extracted from HeLa cells treated for 3 d with siRNAs against DIMT1L or WBSCR22 (, and ). When primer LD2141, which specifically selects the nuclear 21S-C and its precursors (; ), was used to initiate cDNA synthesis on RNAs extracted from DIMT1L-depleted cells, we detected 57% reduction in dimethylation efficiency (). This observation is in agreement with the prediction that DIMT1L is the bona fide 18S rRNA dimethyltransferase. Because the 21S-C is nuclear, we further conclude that in human cells, the modification is synthesized in the nucleus, in contrast to the situation in yeast, in which this modification is reported to occur in the cytoplasm (). RNA extracted from WBSCR22-depleted cells was specifically cleaved at N7G-modified residues and used as template for cDNA synthesis with primer LD2120 or LD2122 (). As expected from the assumption that WBSCR22 is the bona fide 18S rRNA m7G methyltransferase, a substantial 62% reduction in methylation level was observed with both primers. Since m7G1639 is readily detected from primer LD2122, specific to a sequence 3' to the dimethylation site that would stop its extension, we further conclude that 18S rRNA N7G-methylation occurs prior to dimethylation and is thus also nuclear.

rRNA-blocking oligo's decrease rRNA-cDNA synthesis in …

Here we characterized two human methyltransferases with specificity toward the small ribosomal subunit rRNA. We show that their presence in cells, but not their RNA-methylating catalytic activity, is indispensable for pre-rRNA processing steps leading to the synthesis of the 18S rRNA (, , , and and Supplemental Figure S3). We demonstrate that DIMT1L is responsible for N6,N6 dimethylation at positions A1850 and A1851 and that WBSCR22 is required for N7 methylation at nucleotide G1639 ( and ; see also ). These nucleotide positions are equivalent to those modified in yeast by the homologous methyltransferases. We show that WBSCR22 interacts directly with TRMT112, forming a heterodimer, and that the latter protein is required for the metabolic stability of the former (). Taken together, these findings indicate strong conservation of the functions of Dim1/DIMT1L and Bud23-Trm112/WBSCR22-TRMT112 in yeast and human cells (, ; ; ; ; ).

-protein synthesis involves change in language from ..

We tested this prediction by primer extension mapping in cells depleted of DIMT1L or WBSCR22 (). The modification is readily detected by primer extension because it is a bulky modification blocking reverse transcriptase progression during cDNA synthesis, thus generating strong primer extension stops and a “stutter” (). In contrast, detection of m7G by primer extension ideally requires specific cleavage of the RNA at the site of modification. This is achieved by reduction with sodium borohydride followed by aniline-mediated strand scission at the reduced residue (; ).

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