1. Volume 14, Issue 4, Pages 457-464 (May 2004)
Elongator Interactions with Nascent mRNA Revealed by RNA Immunoprecipitation 
Christopher Gilbert, Arnold Kristjuhan, G.Sebastiaan Winkler, Jesper Q Svejstrup 
Molecular Cell 
Volume 14, Issue 4, Pages (May 2004)
DOI: /S (04)

2. Figure 1 Elongator Binds RNA In Vitro
(A) EMSA using Elongator complex and 0.24 fmol radiolabeled 95 b RNA oligonucleotide. The experiments in lanes 2–4 contained 22, 66, and 200 fmol of Elongator complex, respectively. Migration of Elongator-RNA complexes are indicated by lines.
(B) EMSA using Elongator complex, 1 fmol radiolabeled RNA, and cold competitor DNA. The experiments in lanes 2–7 contained 160 fmol of Elongator complex. The experiments in lanes 3–7 contained 10, 20, 56, 159, and 518 fmol of pBluescript plasmid DNA, respectively. Asterisk denotes the migration of the RNA probe, the arrow indicates the position of the wells, and the filled sphere denotes the band-shifted RNA probe.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 RNA Polymerase-RNA Immunoprecipitation
(A) RIP experiment using monoclonal antibodies (4H8) to immunoprecipitate RNAPII from extracts from cells grown in glucose (Glu) and galactose (Gal), respectively. IN, input (crude DNase I-treated extract); IP, immunoprecipitated material.
(B) Control experiment, showing that the amplified DNA products were derived from mRNA not DNA. RT-PCR, polymerase chain reactions containing reverse transcriptase. PCR, polymerase chain reactions without reverse transcriptase.
(C) Control experiment showing that little, if any, crosslinking occurs between epitope-tagged TATA binding protein (TBP-3HA) and RNA.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 Specificity of RNA Immunoprecipitation
(A) RIP experiment using RNAPII-specific monoclonal antibodies (4H8) and anti-Myc antibodies (9E10) to immunoprecipitate RNAPII and RNAPI, respectively, from RPA43-Myc extracts. Quantification is shown beneath the relevant lanes. Note that multiplex RT-PCR reactions including both primer pairs were not possible because 18S rRNA (RDN37) is much more abundant than ARP2 mRNA (see Experimental Procedures for details).
(B) RIP experiment using RNAPII-specific monoclonal antibodies (4H8) and anti-Cbp80 antibodies (Gorlich et al., 1996) to immunoprecipitate RNAPII and CBC, respectively. Relative efficiency of binding to the 5′ end of GAL1 mRNA and RDN37 (rRNA) is shown. Binding to rRNA was set to 1.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
Elongator RIP
RIP experiment using anti-Myc antibodies to immunoprecipitate Elp3-Myc from extracts prepared from cells (ELP3-Myc or nontagged control) grown in glucose and galactose, respectively. IN, input; IP, immunoprecipitated material; RNase + IP, immunoprecipitated material derived from RNase-treated extract. Quantification, adjusted for input, is shown beneath the relevant lanes. The amount in control IPs from cells expressing untagged Elp3 was set to 1.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5 Elongator Associates with Nascent, Unspliced RNA
(A) RIP experiment using anti-Myc antibodies to immunoprecipitate Elp3-Myc and associated nascent, unspliced ARP2 transcripts from extracts prepared from ELP3-Myc or nontagged control cells. IN, input; IP, immunoprecipitated material.
(B) Same as (A), but detection of nascent, unspliced TUB3 transcript. Panels on the right (lanes 5 and 6) depict control experiments showing that the amplified PCR products from Elp3 RIP were indeed derived from mRNA, not DNA. RT-PCR, polymerase chain reactions containing reverse transcriptase; PCR, polymerase chain reactions without reverse transcriptase.
(C) Comparison of the efficiency of crosslinking to nascent ARP2 RNA (“unspliced”) to “spliced” ARP2 RNA. (Upper panel) RT-PCR products derived from the indicated amounts (in μl) of RNA are shown. Note that relatively more input of unspliced RNA than input of spliced RNA is required to give a RT-PCR signal that is similar to the signal derived from the respective Elp3 IPs. (Lower panel) Quantification of upper panel, showing relative amount of unspliced and spliced RNA crosslinked to Elongator (spliced RNA set to 1).
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
Association of Elongator along the Entire Coding Region of an Active Gene
(A) The GAL1 locus and the position of primers used to detect RNA in different regions of the gene. Primer pair A was upstream of the transcriptional start site, and primer pair F was downstream of the GAL1 termination region. Primer pairs B, C, D, and E were all within the transcribed region of GAL1.
(B) Detection of GAL1 RNA isolated from cells grown in glucose and galactose, respectively. The reasons for the varying levels of RNA detected across the gene are not understood but may be due to differences between the primer pairs as well as intrinsic variation in the efficiency of RT-PCR.
(C) Detection of GAL1 RNA isolated from cells grown in galactose after RIP with antibodies (4H8) directed toward RNAPII. Similar results were obtained with a strain expressing flag-tagged Rpb3 (data not shown).
(D) Detection of GAL1 RNA isolated from cells grown in galactose (Elp3-myc and untagged control, respectively) after RIP with 9E11 antibodies. Prior to depiction as graphs, the results in (C) and (D) were corrected for input.
Molecular Cell  , DOI: ( /S (04) )