PMC

(A) Long read sequencing of nascent RNA workflow: Nascent RNA molecules are 3’ end ligated and reverse transcribed. A PCR targeting all cDNAs generates double-stranded cDNAs for long read sequencing.
(B) Long read sequencing observations of S. cerevisiae nascent RNA molecules for six representative genes. Presence (thick line) or absence (thin line) of intron sequence and 3’ end position indicate splicing state and Pol II position, respectively.
(C) Long read sequencing observations of S. pombe nascent RNA molecules is represented as described in (B).

Average interaction force experienced by nascent chain atoms with ERY and the ribosome when we turned on non-bonded parameters one by one residue. (A) Average non-bonded interaction force, per nascent chain atom, between the nascent chain and Erythromycin. (B) Average non-bonded interaction force, per nascent chain atom, between the nascent chains and the ribosome. Error bars represent 95% confidence intervals. Line is to guide the eye and does not represent a model. Note well, the individual trajectory force traces were first aligned such that F_max occurs at time equal to zero, and then the average across trajectories was calculated.

A. 3’ RACE-Seq of hTR in human embryonic stem (hES), HeLa, and UMUC3 cells. hTR transcripts either end at the annotated end of 451 nts or shorter (brown), are >451 nts with genomically templated tails (grey), or are >451 nts with untemplated oligo-A tails (blue). hTR Reads≥200k
B. Nascent RNAend-Seq to enrich nascent RNA tails. 4SU labeled RNA from pulsed cells is conjugated to biotin (Biotin-HPDP, B). Biotin-conjugated nascent RNA is captured with streptavidin (SA) beads and separated from steady state (SS) RNA. Nascent RNA is eluted with DTT, ligated with a 3’ ssDNA adapter, reverse transcribed (RT) and amplified with a gene-specific primer (GSP)
C. RNA bioanalyzer shows 1.7% of 4SU labeled input RNA is recovered vs. 0.04% of 4SU-unlabeled RNA.
D. qRT-PCR for RNA precursors including 47S rRNA and beta-2-microglobulin (B2M) in nascent RNA (blue) and steady state RNA (black). 47S rRNA primers span 5’ externally transcribed spacer (ETS) and B2M primers span an intron-exon junction. Data normalized to exonic GAPDH primers. Error bars represent S.E.M.
E. Sequencing of nascent U2 3’ end with the proportion of reads having a given tail length. Height of the line at templated tail length=0 indicates the proportion of reads with no tail; height of the line at templated tail length=10 indicated the proportion of reads with a tail≤10 nucleotides. Structure of the U2 snRNA transcription unit shown with annotated U2 3’ end graphed at 0 nts and 3’ box at 17 nts. Nascent RNAend-Seq results for U2 nascent (blue line) and steady state (black line) RNA.

(A) Schematic representation of replication forks progressing in both directions through a segment, as detected by the N/A 2D gel electrophoresis technique. We considered two instances in which replication forks progress in both directions through a segment, as detected by either a 5′- or 3′-end probe: (i) replication forks progress towards each other within the examined segment and (ii) replication forks progress in one direction in some cells (one population) and in the opposite direction in other cells (the other population) within the examined segment. These two instances can be discriminated by N/N 2D gel electrophoresis. In the first case, a random or specific termination pattern is expected, while in the second case, no termination is observed. Since we did not detect termination within the segment by N/N 2D gel electrophoresis (Fig. ), only the second instance is shown in the diagram. Two identical segments in two different populations of cells are shown. The positions of the probes (filled rectangles) are shown below the segments. The signals that appeared on the film after hybridization to the specific probes are schematically shown below the specific probes and are labeled a to e as follows: a is the nonreplicated linear segments specifically detected by either the 3′- or 5′-end probe (called the 1N spot), b is the full-length parental strands from which the nascent strands are released in the second dimension at alkaline pH, c is the newly replicated nascent strands of multiple (both large and small) sizes, d is the cross-hybridization signals to the nonreplicating linear molecules, and e is the single-stranded molecules that result from breaks in the linear nonreplicating molecules (from the 1N spot). In the segment on the right, replication forks progress in the 3′-to-5′ direction. In this case, the probe at the 3′ end of the segment detects nascent strands of multiple sizes, while the probe at the 5′ end detects only large nascent strands. In the segment on the left, replication forks move from the 5′ end to the 3′ end of the segment. The probe at the 5′ end detects nascent strands of different sizes, and the 3′-end probe detects only large nascent strands. Since both of these populations of cells are present, both the 3′- and 5′-end probes detect nascent strands of multiple sizes (from different cells). The long diagonal nascent strand arcs detected by these probes are shown at the bottom of the figure. (B) Replication forks progress in both directions through examined segments of the Igh-C region in the early-replicating Igh locus. The examined restriction fragments and the positions of the probes (filled rectangles) are indicated. Both the 5′- and 3′-end probes detected nascent strands of all sizes, demonstrating that replication forks enter the segment from upstream and from downstream. Horizontal arrows below (70Z/3) or above (38C-13) the autoradiograms indicate the direction of replication fork movement. In two regions in the 70Z/3 cell line (μ region and 3′ regulatory region [RR]), the direction of replication fork progression on two allelic chromosomes was examined. Two separate arcs are visible for each allele since the probe detects two different restriction fragments (one from each allele). The difference in the sizes of the segments on the two allelic chromosomes for the 3′ regulatory region is much greater than that for the μ region. Thus, the two nascent strand arcs for the 3′ regulatory region are far apart while the two nascent strand arcs for the μ region are close to each other. Nascent strands of all sizes were detected in all segments after Southern hybridization to either the 5′- or 3′-end probe, indicating that replication forks progress in both directions on both alleles. Twelve fragments were examined by N/N 2D gel electrophoresis, which detects the shapes of the replicated molecules (examples are shown). Only a Y-shaped arc was detected in all segments examined, suggesting that replication forks progress through the fragments in either direction. Abbreviations: E, EcoRI; B, BamHI; H, HindIII; X, XbaI; RFLP, restriction fragment length polymorphism. (C) Schematic representation of replication forks progressing in both directions on two allelic chromosomes detected by N/A 2D gel electrophoresis. This scheme represents the ideal pattern of the autoradiogram one would detect if the replication forks move in both directions through the two allelic chromosomes, which can be distinguished by an RFLP. The two separated nascent strand arcs are indicated by arrows. For the Cμ segment, the smallest nascent strands originating from both chromosomes were not easily detectable, in contrast to what is shown in panel B. In the original autoradiogram, nascent strands smaller than 1.5 kb were weakly detected. This may be due to the fact that the intensities of the signals of the nascent strands from a single allele were weaker (by a factor of 2) than those of strands derived from both alleles simultaneously. In the 3′ regulatory region (B), nascent strands of all sizes originating from the DBA allele were easily detected with the 3′-end probe. However, on the C57BL allele, the smallest nascent strands (up to 800 bp) were not apparent on the autoradiogram since the conditions of N/A 2D gel electrophoresis were not ideal for both segments. Since these two segments (2.5 and 4.0 kb) differ in size by a factor of almost 2, the gel could not be run in a range optimal for both. The 5′-end probe for the HindIII/XbaI segment in the 3′ regulatory region detected nascent strands of all sizes from both alleles, which were apparent on the autoradiogram although they appear very weak in the gels shown in panel B. Since these allelic segments differ in size by only one-third, they can be distinguished by use of the same gel conditions.