Metabolism of small RNAs in cultured human cells

Choudhury, Kanakendu; Choudhury, Indrani; Eliceiri, George L.

doi:10.1002/jcp.1041380227

Cited by 5 publications

(4 citation statements)

References 21 publications

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“…Quantitation by densitometry after silver staining was done under the condition that has given a linear response with the RNA concentration [29]. Relative abundance of each UsnRNA was calculated by normalizing the areas of the peaks with respect to that of corresponding 5S rRNA [28,[32][33][34].…”

Section: Subcellular Fractionation Rna Extraction and Usnrna Fractiomentioning

confidence: 99%

“…Quantitation by densitometry was done following the procedure of Choudhury et al [28] to give a linear response both with X-ray film and the negative of ethidium bromide stained gel. To study the kinetics of labelling of each of the six UsnRNAs during liver regeneration, area of each labelled UsnRNA band was normalised to that of the total unlabelled area of the corresponding 5S rRNA [28,[32][33][34] and plotted against different period of incubation.…”

Section: In Vitro Labelling Of Usnrnas In Isolated Hepatocytesmentioning

confidence: 99%

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Changes in UsnRNA biosynthesis during rat liver regeneration

et al. 1994

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Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50 % upto 48 hrs of RH. followed by gradual increase to reach the normal level within one month of R H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of RH. but in 24 and 72 hrs of RH. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of RH. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30-50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of RH. Thus, it may be concluded that the changes in UsnRNAs' level during th e proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver. (Mol Cell Biochem 141: 71-77, 1994).

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Section: Subcellular Fractionation Rna Extraction and Usnrna Fractiomentioning

confidence: 99%

Section: In Vitro Labelling Of Usnrnas In Isolated Hepatocytesmentioning

confidence: 99%

Changes in UsnRNA biosynthesis during rat liver regeneration

et al. 1994

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“…Subsequent studies revealed that there are relatively few intermediate bands between these molecules shortened by three or four nucleotides and the mature-length U2 RNA (3,27), and this was also the case when the 3Ј processing of human pre-U2 RNA in Xenopus oocytes was investigated (33). It should be noted, as regards the in vivo situation, that the presence of Sm proteins at nucleotides 100 to 104 might be expected to favor a singlestranded conformation of the 3Ј-terminal nucleotides of pre-U2 RNA (i.e., nucleotides 194 to 199).…”

Section: Fig 8 Substrate Competitionmentioning

confidence: 99%

3′ Processing of Human Pre-U2 Small Nuclear RNA: a Base-Pairing Interaction between the 3′ Extension of the Precursor and an Internal Region

Huang

Jacobson

Pederson

1997

Molecular and Cellular Biology

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The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3 ends. The 3 processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3 processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3 ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3 end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3 processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3 extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3 extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3 processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3 processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3 extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3 processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3 processing of human U2 RNA.Small nuclear RNAs (snRNAs) are essential components of ribonucleoprotein particles that are involved in pre-mRNA processing (1, 16). The biosynthetic maturation pathway of the spliceosomal snRNAs involves many steps in both the nucleus and cytoplasm (20,35). Except for U6 (14,26), the snRNAs of the major class of spliceosomes are transcribed by RNA polymerase II. Once transcribed, pre-U1, -U2, -U4, and -U5 snRNAs carrying 3Ј extensions of various lengths are rapidly transported to the cytoplasm, where they undergo small nuclear ribonucleoprotein (snRNP) assembly, 5Ј-cap hypermethylation, and 3Ј processing before being imported back into the nucleus (3, 4, 6, 8, 13, 17-20, 25, 32, 34, 35).U2 RNA is the second most abundant snRNA in mammalian cell nuclei (25). Pre-U2 RNA molecules having 3Ј extensions of 10 to 16 nucleotides have been detected in both rat liver (29) and HeLa cells (7,32). We have previously shown that pre-U2 RNA 3Ј processing occurs accurately in several in vitro systems of progressive refinement, including HeLa cell whole cytoplasmic extract (32), an ammonium sulfate fraction of a HeLa cell cytoplasmic S100 fraction (12), and a 15S glycerol gradient fraction of the latter (9, 12). Because this 15S system is free of a nonspecific ribonuclease present in the former preparations, it has enabled us to study pre-U2 RNA 3Ј processing in considerable detail (9). In co...

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“…Moreover, 3Ј end formation appears to be an RNA processing event, because U2 (but apparently not U1) transcription continues for up to 800 nucleotides (nt) beyond the 3Ј box (15,47). Over the next 90 min (12,51), the processed intermediate is exported to the cytoplasm, where the mature 3Ј end is generated by 3Ј trimming (21,29), the 5Ј monomethyl cap is trimethylated, Sm proteins are assembled onto the snRNA (44,45), and the nearly mature small nuclear ribonucleoprotein particle is imported back into the nucleus (31) where it undergoes further base modifications (17,71) before it can function in RNA processing. Little is known about either the nuclear or cytoplasmic 3Ј processing events, but the dependence of efficient 3Ј-box-directed processing on a PSE-bearing snRNA promoter links an early step at the promoter to later events at the 3Ј end of the gene, perhaps through specific modification of the polymerase and/or recruitment of factors that travel along with it.…”

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confidence: 99%

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Jacobs

Ogiwara

Weiner

2004

Molecular and Cellular Biology

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U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3 processing is directed by a highly conserved 3 end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3 processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3 end formation.RNAs that encode proteins are transcribed by RNA polymerase II (Pol II) in almost all eukaryotes. In contrast, untranslated RNAs are transcribed by all three RNA polymerases: 5.8, 18, and 28S rRNA by Pol I; 5S rRNA, tRNA, and U6 small nuclear RNA (snRNA) by Pol III (56); and the other U snRNAs, which function in mRNA splicing and various RNA processing events, by Pol II (27). Kinetoplastid protozoa, a class of early diverging eukaryotes, are exceptions to these rules. Kinetoplastid snRNAs are transcribed not by Pol II but by Pol III (65), and certain mRNAs, including the immunologically important variant surface glycoprotein message, are hybrids of a U snRNA-like spliced leader transcribed by Pol II and a protein-coding mRNA body transcribed by Pol I (19).Although U snRNAs and mRNAs are both transcribed by Pol II in mammals, the genes are very different. U snRNA promoters have no TATA box and rely instead upon a dedicated U snRNA-specific promoter consisting of a highly conserved proximal sequence element (PSE) and an enhancer-like distal sequence element spaced one nucleosome apart (27). In addition, U snRNA genes are short (typically only a few hundred base pairs) and lack introns, whereas genes encoding mRNAs can span megabases and usually contain many introns. Also, U snRNA genes are typically present in multiple copies in higher eukaryotes-the human U1 and U2 genes are tandemly repeated (6, 40, 66, 68)-whereas most protein-coding genes are present in only one or a few copies per haploid genome.U snRNA processing also differs from mRNA processing. U snRNAs are neither spliced nor polyadenylated; instead, formation of the first U snRNA interme...

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Metabolism of small RNAs in cultured human cells

Cited by 5 publications

References 21 publications

Changes in UsnRNA biosynthesis during rat liver regeneration

Changes in UsnRNA biosynthesis during rat liver regeneration

3′ Processing of Human Pre-U2 Small Nuclear RNA: a Base-Pairing Interaction between the 3′ Extension of the Precursor and an Internal Region

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

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